Category Archives: Stem Cells

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Global Synthetic Stem Cells Market Advancements and Outlook 2020 to 2027 North Carolina State University and Zhengzhou University and others

The prominent players in the market are North Carolina State University and Zhengzhou University

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Global Synthetic Stem Cells Market Advancements and Outlook 2020 to 2027 North Carolina State University and Zhengzhou University and others KSU |...

Clinical trial data supporting the safety of the CRISPR-Cas9 genomic editing tool was presented on Monday by Intellia Therapeutics (NASDAQ:NTLA) for its lead product, NTLA-2001. The data was highly encouraging. However, despite NTLA-2001's positive early results as a potential treatment for the rare disease transthyretin (TTR) amyloidosis, there's still a long way to go before Intellia could bring it to market.

In transthyretin amyloidosis, cells in the liver produce misfolded TTR proteins, which accumulate throughout the body, causing debilitating complications that can involve the digestive system, nervous system, and heart. Once symptoms appear, they grow progressively worse, and the disease leads to death within a median of 4 to 17 years among patients with nervous system involvement, and 2 to 6 years among patients with cardiac involvement.

NTLA-2001 edits the genes in those liver cells, removing the segment that produces those lethal misfolded proteins.

Worldwide, an estimated 250,000-550,000 people suffer from some form of amyloidosis.

IMAGE SOURCE: GETTY IMAGES.

An interim readout from Intellia's ongoing phase 1 trial found that a single high-dose infusion of NTLA-2001 led to an 87% mean reduction in the amount of misfolded TTR in patients' bloodstreams, with a maximum reduction of 96% by day 28 in one patient. Encouragingly, no serious adverse events were observed in the six study participants. While this is a small pilot study, in previous studies of NTLA-2001 in mice, the maximum reductions in TTR persisted for 12 months after a single treatment.

All of this data provides an early indication that CRISPR gene therapies are safe and efficacious as treatments for at least some genetic diseases.

There are other treatments on the market for TTR amyloidosis, but one thing that would set CRISPR apart is the relative simplicity of administering it. And that factor could lead insurers to favor CRISPR treatments for certain rare and debilitating diseases such as TTR amyloidosis and hemophilia.

For example, Alnylam's (NASDAQ:ALNY) RNA-silencing therapy Onpattro requires an infusion every three weeks at a clinician's office. Ionis Pharmaceuticals' (NASDAQ:IONS) Tegsedi requires regular injections, though they can be self-administered. Both are priced in the neighborhood of $345,000 per year, and Onpattro comes with the additional costs associated with going to a medical office and having an infusion set up. Then there is Pfizer's (NYSE:PFE) once-daily oral medication Vyndamax, which costs $225,000 annually.

As a one-time infusion, gene therapy may become a compelling option for both patients and insurers, particularly given the high prices of currently available treatments. Though TTR amyloidosis treatments are a niche market, in 2020, Onpattro generated sales of $306 million, Tegsedi just under $70 million, and Vyndamax $429 million. Assuming that Intellia charges more for NTLA-2001 -- a one-time treatment with bluebird bio's (NASDAQ:BLUE) gene therapy for beta-thalassemia, Zynteglo, costs about $1.8 million -- TTR amyloidosis treatment could easily become a multibillion-dollar addressable market for the biotech.

Notably, CRISPR therapy for TTR amyloidosis may also put less stress on the healthcare system than the lentivirus and adenovirus gene therapies that are further along in clinical trials. Consider, for instance, Zynteglo, which requires a significant amount of effort and processing prior to treatment. First, physicians must extract stem cells from the patient, which must then be transported to and treated by bluebird bio. In the meantime, the patient undergoes "myeloablative conditioning" -- essentially knocking down the patient's bone marrow in preparation for a transplant of the edited stem cells, which will contain a repaired version of the gene that (when mutated) causes beta-thalassemia. This complicated process requires treatment at a qualified transplant center.

By comparison, for TTR amyloidosis, NTLA-2001 requires pre-medication with steroids and antihistamines. That's it. No prolonged patient preparation at the hospital. No bone marrow suppression. No shipping the patient's stem cells to a lab. The relative simplicity of administering CRISPR therapies is just one reason for the degree of excitement they are generating.

It may also give them a lower total cost of treatment than current gene therapies, which could make these therapies more palatable to insurers. If NTLA-2001 pans out, we may see a new biotech boom, with Intellia leading the charge.

Before investors get their hopes up too much, remember that these results were from a six-person, phase 1 trial, and that Intellia now holds a market cap of roughly $11 billion. In fact, its valuation rose by about $2.8 billion in a single trading session after the interim trial data was made public. That gain was more than the current $2.1 billion market cap of bluebird bio, which already has an approved gene therapy on the market as well as a CAR-T therapy, and has two more candidates in phase 3 trials.

For further context, bluebird bio announced phase 1 results for Zynteglo in December 2014. While Zynteglo was approved for use in the EU in late 2019, bluebird bio faced some backlash on pricing, and the company isn't selling it in Germany because the two sides could not agree on pricing.

Moreover, the NTLA-2001 study excluded patients who had previously received RNA-silencing therapy, and none of these patients had previously taken Vyndamax either. How previous treatments will affect the way patients respond to NTLA-2001 is not yet known. And with hundreds of millions of dollars in revenue annually on the line, it is doubtful that Alynam, Ionis, or Pfizer will surrender this market without a fight.

In sum, Intellia will still need to conduct several years of trials, leap many regulatory hurdles, and outmaneuver an array of rivals stand before it can declare the CRISPR-Cas9 platform a winner. Not only that, but -- recognizing that future studies won't be cheap -- Intellia has already proposed another public offering of $400 million worth of common stock this week, diluting its current shareholders.

So while long-term Intellia shareholders have reason to celebrate, let bluebird bio serve as a cautionary tale. That biotech was once flying high on positive trial data, hitting a market cap of around $15.5 billion in March 2018. Since then, its shares have nose-dived by more than 80%. This despite the fact that it now has two approved therapies and two more candidates in phase 3 trials.

As such, I would be concerned about investing new money in Intellia now. I suspect it will soon reach its peak for the foreseeable future. Biotech investing can be gut-wrenchingly fickle, and investors may want to consider taking a basket approach to high-risk clinical-stage biotechs, rather than investing too heavily in a single player.

This article represents the opinion of the writer, who may disagree with the official recommendation position of a Motley Fool premium advisory service. Were motley! Questioning an investing thesis -- even one of our own -- helps us all think critically about investing and make decisions that help us become smarter, happier, and richer.

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2 Reasons to Buy Intellia -- and 1 Big Reason Why I Won't - The Motley Fool

Katie and Jacob Lamprecht are doing what they can to buy more time for their three children, all of whom have a rare degenerative illness that has no cure.

Last fall, 10-year-old Kiara Lamprecht and her eight-year-old sister Hannah were diagnosed with juvenile neuronal ceroid lipofuscinoses also known as CLN3 or juvenile Batten disease.

Then five-year-old AJ, theirhalf-brother,was also diagnosed with the illness last month.

As the parents scour research seeking potential treatments, fundraisers are underwayto allow the family to afford them.

"Time is of the essence. We don't have years and years and years to figure this out," said Katie Lamprecht from the family's home in Evansburg, Alta.,about 90 kilometres west of Edmonton.

Batten disease, an inherited and fatal genetic illness, has various forms. In this case, it's caused by a mutation that results in cells not producing enough of the CLN3 protein that helps clear waste products. Over time, toxins build up and brain cells start to die.

Symptoms usually start showing in children aged four to seven. The first is often vision loss, followed by changes in cognition and behaviour, seizures, then declining motor skills.

Those with the disease will die in their teens or 20s.

CLN3 is a rare condition in Canada, according toStphane Lefranois, researcher professor at the Institut National de la Recherche Scientifique in Quebec.

Juvenile Batten disease develops when a child inherits two copies of the genetic mutation from their parents, each of whom would be carrying one defective gene. Because two defective copies are needed for Batten disease to develop, the parents would not be affected, said Lefranois.

The mutation's prevalence varies throughout the world. In some regions,it shows up in one of 12,000 live births; in others, it's found in one of 100,000 births, he said.

It's"quite rare" that sisters Kiara and Hannah were diagnosed with the disease,said Lefranois, but theodds of AJ's diagnosisgiven he has a different motherare "astronomically low."

Jacob Lamprecht moved to another part of the world andmarried Katie, an unknowing carrier,and AJ inherited both mutations. The Lamprechtshave been told the chance of their situation occurring is about one in eight billion.

According to the parents, both girls are legally blind. AJ's eyesight is worsening but he hasn't had a vision test yet.Hannah has developed behavioural issues, with episodes of kicking, screaming and biting.Abrain scan conducted last year shows she has sufferedsome brain damage and is experiencing seizures, said her father.

So far, over $113,500 has been raised for potential treatments. But the parents are unsure about their next move.

"There's nothing concrete that we can follow," said Katie Lamprecht.

There is currently no cure for CLN3, so most treatment just manages the symptomslike medications for seizures or Braille instruction for children losing their vision, said Dr. Jonathan Mink, CLN3 researcher and a professor of pediatric neurology at New York's University of Rochester.

But clinical trials in the United States some completed, some ongoing are makingheadway, he said.

Among them are clinical trials for gene therapy, a technique that modifies a person's faulty genes to help cells function normally.

"The hope is, particularly with gene therapy, that we could give something to a very young child and that would correct the problem and they never have any symptoms," said Mink.

The Lamprechts are looking at gene therapy, which is deemed an experimental treatment option by Health Canada, as well as a stem cell operation. The latter is a last resort, they said, requiringtravelto the U.S. and a cost ofabout $1.8 million per child.

Some people have undergone such operations, but Mink would advise against it because there aren't many published results and, through his clinical practice, he hasn't seen any significant improvement in patients.

Undergoing a stem cell operation might also disqualify a child from future clinical trials or treatments, he added.

After the initial diagnosis, families often go through the stages of grief before focusing on giving their child the best possible quality of life for their remaining years, said Mink.

Families who have more than one child with the disease feel a greater impact, but Mink and his team are "so impressed by how resilient people are."

Jacob and Katie Lamprecht are among the resilient, savouring moments with their children while focusing on how to save them.

COVID-19, for example, became an opportunity for Katie Lamprechtto embracefull-time homeschooling, lettingher be with them every day.

Kiara has been approved for Make-A-Wish Canada, so the family is debating whether to visit SeaWorld or Disney World.

"We're not just sitting around crying all the time," said Katie.

"Most of the time I feel like we're going to beat this."

Excerpt from:
'Time is of the essence': Alberta family seeking treatment for 3 kids with rare, degenerative illness - CBC.ca

This article was originally published here

J Med Chem. 2021 Jun 4. doi: 10.1021/acs.jmedchem.0c02194. Online ahead of print.

ABSTRACT

A 3-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

PMID:34085825 | DOI:10.1021/acs.jmedchem.0c02194

Originally posted here:
Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro - DocWire News

Abstract

Hepatocellular carcinoma (HCC) is driven by repeated rounds of inflammation, leading to fibrosis, cirrhosis, and, ultimately, cancer. A critical step in HCC formation is the transition from fibrosis to cirrhosis, which is associated with a change in the liver parenchyma called ductular reaction. Here, we report a genetically engineered mouse model of HCC driven by loss of macroautophagy and hemizygosity of phosphatase and tensin homolog, which develops HCC involving ductular reaction. We show through lineage tracing that, following loss of autophagy, mature hepatocytes dedifferentiate into biliary-like liver progenitor cells (ductular reaction), giving rise to HCC. Furthermore, this change is associated with deregulation of yes-associated protein and transcriptional coactivator with PDZ-binding motif transcription factors, and the combined, but not individual, deletion of these factors completely reverses the dedifferentiation capacity and tumorigenesis. These findings therefore increase our understanding of the cell of origin of HCC development and highlight new potential points for therapeutic intervention.

Liver cancer is predicted to be the third leading cause of cancer-related deaths by 2030 (1). Hepatocellular carcinoma (HCC) is the major form of liver cancer and develops in patients with chronic liver conditions, including viral hepatitis, as well as alcoholic and nonalcoholic fatty liver disease (2). Generally, chronic liver injuries lead to inflammation, stromal activation, regeneration, fibrosis, and cirrhosis before progression to HCC (3).

Autophagy (strictly macroautophagy but hereafter referred to simply as autophagy) is a catabolic membrane-trafficking process that serves to deliver cellular constituents including misfolded proteins and damaged organelles to lysosomes for degradation (4). There is now clear evidence that autophagy is important in various diseases including neurodegenerative diseases, chronic liver diseases, and cancer (57). The role of autophagy in cancer, however, is complex and not fully understood, with seemingly opposing roles described in different tumors and at different stages of tumor evolution (812). In the early stages of malignant transformation, autophagy removes damaged mitochondria responsible for the production of reactive oxygen species (ROS) (13) and prevents genomic instability (14), highlighting its role in preventing tumor initiation. Conversely, in established tumors, autophagy not only can adopt a protumorigenic role, for example, by promoting survival under hypoxic conditions (15) and supporting invasion and metastasis (16), but also can have a tumor-suppressive role by preventing the proliferative outgrowth of disseminated tumor cells from dormant states at metastatic sites (1719).

In the liver, autophagy has primarily been described as tumor suppressive (11). Liver-specific deletion of the central autophagy-related protein 5 (ATG5) or ATG7 in mice leads to the formation of liver steatosis, inflammation, ROS production, oval cell formation, fibrosis, hepatomegaly, and the development of HCCs (11, 20). In many cases, loss of autophagy causes accumulation of the autophagy adapter protein p62 (Sqstm1), and this can influence antioxidant responses by affecting the axis between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) (21). In autophagy-deficient livers, studies have shown that p62 accumulation activates the NRF2 signaling pathway to induce metabolic reprogramming, hepatomegaly, and tumorigenesis (22, 23).

The liver is a plastic organ in which cell fate can change upon injuries to regenerate liver function loss. Hepatocytes and cholangiocytes, epithelial cells that form the liver parenchyma and the bile duct, respectively, can transdifferentiate into one another to reestablish bile duct or liver parenchyma functions (24, 25), with hepatocytes being the primary source of liver regeneration upon injury. Following chronic injury, ductular cells develop in the liver parenchyma when hepatocyte or cholangiocyte function is severely impaired, a process called ductular reaction (26). The ductular reaction is a repair mechanism for generating new hepatocytes or cholangiocytes, depending on which liver cells are injured (27). However, the origin of the ductular reaction and its role in liver tumorigenesis are controversial with reports indicating that ductular cells can arise from cholangiocyte expansion (28, 29) or through hepatocyte dedifferentiation (30, 31) and reports concluding that the ductular reaction is involved in forming HCC (32, 33), while other studies report the opposite (34, 35). Autophagy-deficient livers undergo a ductular reaction (36), and we considered this as an excellent system in which to explore its origin and the role, this phenomenon plays in tumorigenesis.

In this study, we report that autophagy prevents hepatocyte dedifferentiation into ductular liver progenitor cells (LPCs). This ductular LPC population affects HCC formation in autophagy-deficient livers. Mechanistically, we show that autophagy deletion activates both yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes, which are connected to the ductular reaction leading, ultimately, to tumorigenesis. We show that YAP/TAZ coexpression is required to trigger the ductular reaction and tumorigenesis in autophagy-deficient livers.

Autophagy loss in the murine liver results in hepatomegaly, inflammation, and fibrosis leading to the formation of liver HCCs at 12 months of age (20). Phosphatase and tensin homolog (PTEN) expression is lost in approximately half of human liver cancers, and hepatic Pten-deficient mice develop HCC at 74 weeks (37). To accelerate the autophagy phenotype in the liver, we used the liver-specific promoter Albumin-Cre to selectively delete either Atg7flox/flox or Atg5flox/flox in the liver in combination with either heterozygous Pten+/flox (Alb-Cre+; Atg7fl/fl; Pten+/fl or Alb-Cre+; Atg5fl/fl; Pten+/fl) or homozygous Ptenflox/flox (Alb-Cre+; Atg7fl/fl; Ptenfl/fl or Alb-Cre+; Atg5fl/fl; Ptenfl/fl). The reduced gene dosage of Pten in an autophagy-deficient background significantly decreased mouse life span similarly in males and females (Fig. 1A and fig. S1A). At end point, while Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg5fl/fl; Pten+/fl mice developed liver HCCs (Fig. 1B and fig. S1B), Alb-Cre+; Atg7fl/fl; Ptenfl/fl and Alb-Cre+; Atg5fl/fl; Ptenfl/fl mice were culled because of extensive hepatomegaly and did not form tumors. To evaluate whether the decreased survival of Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg5fl/fl; Pten+/fl mice was a result of an early tumor onset, we compared the tumorigenesis of Pten+/+ and Pten+/fl mice with an autophagy-deficient background at 140 days. This revealed that heterozygous deletion of Pten significantly accelerated tumorigenesis in autophagy-deficient livers (Fig. 1, B and C, and fig. S1, B and C). Although conditional double knockout mice did not develop HCC at end point (4 to 5 weeks), they presented with excessive liver overgrowth. When we compared the liver size in 4- to 5-week-old mice, we observed that PTEN loss significantly increased the hepatomegaly of autophagy-deficient livers (Fig. 1D and fig. S1D).

(A) Kaplan-Meier analysis comparing overall survival of mice between males and females (left), males only (middle), or females only (right) (n = 6 males and n = 7 females per group). Data were analyzed by log-rank Mantel Cox test (***P < 0.001 and ****P < 0.0001). (B) Macroscopic pictures from a representative Alb-Cre+; Atg7fl/fl (Alb-Cre+; 7fl/fl) (top) and Alb-Cre+; Atg7fl/fl; Pten+/fl (Alb-Cre+; 7fl/fl; P+/fl) (bottom) liver in 140-day-old mice. (C) Quantification of tumor numbers in Alb-Cre+; 7fl/fl and Alb-Cre+; 7fl/fl; P+/fl at 140 days. Data are means SD of six mice per group and were analyzed by Mann-Whitney test (**P < 0.01). (D) Liver-to-body weight ratio in 4- to 5-week-old mice. Data are means SD of five mice per group and were analyzed by one-way analysis of variance (ANOVA) with Tukey correction for multiple comparison tests (***P < 0.001 and ****P < 0.0001). Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S1D. (E) Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis of neutrophil recruitment (Ly6G), hepatic stellate cell activation (-SMA), and collagen deposition (Sirius Red) on paraffin-embedded sections of livers from 4- to 5-week-old mice. Red arrowhead represents ductular structures. Scale bars, 50 m. Left: Representative staining. Right: Quantifications. Data are means SD of four or five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, **P < 0.01, and ****P < 0.0001). All data points are the mean from five pictures per mouse. FoV, field of vision. Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S1 (E to G).

Next, we assessed whether PTEN loss promotes early development of a tumor-permissive microenvironment in 4- to 5-week-old autophagy-deficient livers by looking for markers of inflammation (38) and fibrosis. This showed that both hemizygous and homozygous Pten deletion significantly increased the recruitment of Ly6G+ neutrophils (Fig. 1E and fig. S1E) and activated smooth muscle actin+ (-SMA+) expressing hepatic stellate cells (Fig. 1E and fig. S1F) in the parenchyma of autophagy-deficient livers, concomitant with a significantly enhanced collagen deposition (Fig. 1E and fig. S1G). PTEN deficiency in 4- to 5-week-old autophagy-competent livers (Alb-Cre+; Pfl/fl) did not result in hepatomegaly, inflammation, hepatic stellate cell activation, or fibrosis (Fig. 1, D and E, and fig. S1, D to G). Together, our data suggest that PTEN loss accelerates the early formation of a tumor-prone microenvironment (inflammation, hepatic stellate cell activation, and fibrosis) and tumorigenesis in autophagy-deficient livers.

Following histological examination, we observed an accumulation of atypical ductular structures in the parenchyma of conditional double knockout livers (Fig. 1E), called ductular reaction. Under normal conditions, the liver has ductular structures, called the bile duct, that are formed out of cholangiocytes (Fig. 1E). The ductular reaction is a regeneration program that occurs in the liver following chronic liver injury that impairs the hepatocyte capacity to regenerate the liver (27). To evaluate whether hepatocytes are injured upon loss of autophagy, we first assessed the expression of enzymes for liver damage in the serum of 4- to 5-week-old livers. All autophagy-deficient livers had a significant increase in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and -glutamyl transferase (GGT) levels in comparison to wild-type (WT) (Alb-Cre+; Atg7+/+ or Atg5+/+; Pten+/+) mice (Fig. 2A and fig. S2, A to D). In addition, we determined whether hepatocytes were dying in our model by looking for cells positive for cleaved caspase 3 (CC3), a marker of apoptosis. We noted a significant augmentation of CC3+ hepatocytes in 4- to 5-week-old autophagy-deficient livers when compared to WT livers (Fig. 2, B and C, and fig. S2E), indicating that autophagy prevents hepatocyte cell death. Next, we observed a significant accumulation of the ductular markers sex-determining region Y-box 9 (SOX9), cytokeratin-19 (CK19), and panCK in Alb-Cre+; Atg7fl/fl; Ptenfl/fl or Alb-Cre+; Atg5fl/fl; Ptenfl/fl livers in comparison to Alb-Cre+; Atg7fl/fl; or Alb-Cre+; Atg5fl/fl single knockout counterparts (Fig. 2, B and D to F, and fig. S2, F to H), confirming that the ductular reaction is occurring in our accelerated model.

(A) Serum analysis of the liver damage markers ALP, ALT, AST, and GGT levels in 4- to 5-week-old mice. Data are means SD of three to five mice per group and were analyzed by one-way ANOVA with Dunnett correction for multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S2 (A to D). (B) IHC analysis of cell death (CC3) and the duct markers SOX9, CK19, and panCK on paraffin-embedded sections of livers from 4- to 5-week-old mice. Scale bars, 50 m. (C to F) Quantification of CC3 (C), SOX9 (D), CK19 (E), and panCK (F) from (B). Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (**P < 0.01, ***P < 0.001, and ****P < 0.0001). All data points are the mean from five pictures per mouse. Please note data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S2 (E to H).

As the ductular reaction is a regenerative process for the de novo generation of hepatocytes upon chronic liver injury (2831), we hypothesized that ductular cells in our model are LPCs forming to repair injured hepatocytes. To test this, we first looked at the expression of liver stem cell markers in Atg- and Pten-deficient livers and found increased levels of epithelial cell adhesion molecule (EpCAM), CD133, and CD44 within ductular cells (Fig. 3A and fig. S3, A to C) of autophagy-deficient livers. The expression of the stem cell makers was autophagy dependent but PTEN independent (Fig. 3A and fig. S3, A to C), although Pten deletion appears to exacerbate the phenotype caused by Atg5 or Atg7 deletion. In addition, we assessed the expression of a-fetoprotein (AFP), a fetal marker reexpressed during HCC and liver stem cell regeneration (39). We observed a significant increase in Afp mRNA levels (Fig. 3B and fig. S3D) and AFP protein level in the serum (Fig. 3C and fig. S3E) of autophagy-deficient mice when compared to WT counterparts.

(A) IHC analysis of the liver stem cell markers EpCAM, CD133, and CD44 on paraffin-embedded sections of livers from 4- to 5-week old mice. Left: Representative staining. Scale bars, 50 m. Right: Quantifications. Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). All data points are the mean were from five pictures per mouse. Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S3 (A to C). (B) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of Afp mRNA isolated from 4- to 5-week-old livers. 18S was used as the internal amplification control. Data are means SD of three mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (**P < 0.01 and ****P < 0.0001). All data points are the mean from technical triplicates. CT, cycle threshold. (C) Enzyme-linked immunosorbent assay (ELISA) analysis of AFP from the serum of 4- to 5-week-old mice. Data are means SD of three mice per group and were analyzed by one-way ANOVA with Dunnett correction for multiple comparison tests (****P < 0.0001). All data points are the mean from technical triplicates. (D) Schematic representation of the lineage tracing experiment for ductular origin. Eight-week-old Atg7flox/flox; Ptenflox/flox; Rosa26mTmG/mTmG mice were infected with hepatocyte-specific Cre-expressing adenovirus (AAV8-TBG-Cre) and aged for 40 days. Rosa26mTmG, Rosa26LoxP-Tomato-Stop-LoxP-GFP. (E) Representative IHC analysis of GFP, tdTomato and SOX9 staining on paraffin-embedded serial sections of liver from Atg7flox/flox; Ptenflox/flox; Rosa26mTmG/mTmG mice 40 days after infection with AAV8-Cre or the vehicle control (AAV8-null). Scale bars, 20 m.

We were interested to know how the ductular-reactive cells were forming within the liver parenchyma. It has been established that ductular-reactive cells can originate from dedifferentiated hepatocytes in the parenchyma (30, 31) or from the activation and the proliferation of hepatic progenitor cells from the canal of Hering to regenerate the liver parenchyma when the regenerative function of hepatocytes is impaired (29). To determine the cell of origin for the ductular-reactive cells in our model, we crossed Alb-Cre; Atg7fl/fl; Ptenfl/fl or Alb-Cre; Atg5fl/fl; Ptenfl/fl mice with the double reporter Rosa26LoxP-Tomato-LoxP-GFP (Rosa26mTmG) and caused Cre-mediated recombination only in hepatocytes using the AAV8-TBG-Cre adeno-associated virus (AAV) (Fig. 3D and fig. S3F), where the Cre recombinase is expressed under the hepatocyte-specific thyroxine binding globulin (TBG) promoter (29). Following recombination, green fluorescent protein (GFP) will only be expressed in hepatocytes at the membrane, while non-recombined cells and unaffected tissues will remain Tomato+. Using this approach, we found that SOX9+ ductular-reactive cells expressed GFP at the membrane 40 days following AAV8-Cre infection in autophagy-deficient livers (Fig. 3E), confirming the hepatocyte origin of the ducts (fig. S3G). Together, our data establish that autophagy prevents dedifferentiation of hepatocytes into ductular LPCs.

ATG7-deficient livers develop HCCs at around 1 year of age (20). Since the ductular reaction is an early event following autophagy inhibition to regenerate the liver and ductular reactive cells express stem cell markers (Fig. 3A and fig. S3, A to C) found in cancer stem cells from HCC (40), we hypothesized that ductular LPCs form HCCs in autophagy-deficient livers. To test this, we first assessed whether autophagy-deficient HCCs retain the expression of the duct marker SOX9, and we noted the presence of two distinct hepatocyte populations (SOX9+ and SOX9) in the normal region surrounding liver HCCs, with SOX9+ hepatocytes found adjacent to ductular structures (Fig. 4A). We found that hepatocytes forming HCCs preserved the ductular marker SOX9 (Fig. 4A). To further evaluate the role of the ductular reaction in tumorigenesis, we infected Alb-Cre+; Atg7fl/fl; Pten+/fl and WT mice with the AAV8-TBG-GFP adenovirus at 6 weeks of age to label hepatocytes with GFP (Fig. 4B). At this age, the ductular reaction is occurring in autophagy-deficient livers, which allows us to distinguish and discriminate between resident hepatocytes (GFP+) and ductular reactive cells (GFP) following AAV8-TBG-GFP infection to trace their role in tumorigenesis. First, we confirmed that at 7 days after AAV8-TBG-GFP infection, SOX9+ LPCs were GFP, while hepatocytes (SOX9) expressed GFP in autophagy-deficient livers (Fig. 4C), confirming that ductular LPCs are not expressing GFP following AAV8-TBG-GFP infection. We then assessed the expression of GFP in autophagy-deficient HCCs 100 days after AAV8-TBG-GFP infection. This revealed that tumors forming in Alb-Cre+; Atg7fl/fl; Pten+/fl livers expressed no GFP in comparison to the surrounding normal hepatocytes, which retained GFP expression (Fig. 4D), highlighting that the ductular cells initiate tumorigenesis in autophagy-deficient livers. We also found that high expression of SOX9 correlates with a decreased survival in human HCCs (Fig. 4E). Together, our data establish that ductular LPCs, formed early upon autophagy deficiency, ultimately lead to the generation of HCCs in autophagy-deficient livers.

(A) IHC analysis of the duct marker SOX9 on Alb-Cre+; Atg7fl/fl; Pten+/fl livers from 140-day-old mice. The red dashed line separates tumor (T) from normal tissue (NT) in the liver. Red and green rectangles outline SOX9+ and SOX9 region in normal tissue, respectively. Scale bar, 100 m. (B) Schematic representation of lineage tracing for tumor origin. Six-week-old Alb-Cre+; Atg7fl/fl; Pten+/fl and WT mice were infected with hepatocyte-specific GFP-expressing adenovirus (AAV8-TBG-GFP) and aged for either 7 or 100 days. (C) Immunofluorescence (IF) analysis of GFP and SOX9 on Alb-Cre+; Atg7fl/fl; Pten+/fl and WT livers 7 days following AAV8-TBG-GFP infection. 4,6-diamidino-2-phenylindole (DAPI) stains nuclei. Scale bars, 75 m. (D) IHC analysis of GFP on Alb-Cre+; Atg7fl/fl; Pten+/fl or WT livers 100 days following AAV8-TBG-GFP infection. The red dashed line separates tumor from normal tissue in the liver. Scale bars, 100 m. (E) Kaplan-Meier analysis comparing overall survival between high and low SOX9 mRNA expression in human liver cancer data (The Cancer Genome Atlas Liver Hepatocellular Carcinoma). Each group represents 20th lower and 20th higher percentile (n = 72 per group).

Blocking the formation of the ductular reaction would be beneficial in preventing human HCC (41). YAP and TAZ are transcriptional coactivators essential in controlling organ size (42), hepatocyte dedifferentiation (31), stemness (43), and liver tumorigenesis (44, 45). The Hippo pathway regulates the activation of YAP and TAZ, and phosphorylation of both coactivators primes them for degradation. As our autophagy-deficient liver model develops severe hepatomegaly (Fig. 1D and fig. S1D), dedifferentiates hepatocytes into ductular LPCs (Figs. 2 and 3 and figs. S2 and S3), and induces tumorigenesis, we next investigated whether YAP and TAZ are active in early-stage autophagy-deficient livers exhibiting ductular reaction. First, we compared the protein levels of the inactive forms of YAP and TAZ (phosphorylated YAP and phosphorylated TAZ), with the levels of total YAP and total TAZ (active forms) in 4- to 5-week-old livers (Fig. 5A). We noticed that the ratio of phosphorylated YAP and phosphorylated TAZ was reduced in autophagy-deficient livers in comparison to WT counterparts (Fig. 5A), highlighting that unphosphorylated YAP and unphosphorylated TAZ accumulate in autophagy-deficient livers undergoing ductular reaction.

(A) Immunoblotting analysis of phosphorylated YAP (p-YAP), total YAP, phosphorylated TAZ (p-TAZ), total TAZ, CTGF, ATG7, and PTEN from 4- to 5-week-old total liver lysates. Extracellular signalregulated kinase 2 (ERK2) was used as the loading control. (B) Quantitative RT-PCR analysis of the YAP/TAZ targets Ctgf, Cyr61, and Areg mRNA isolated from 4- to 5-week-old livers. 18S was used as the internal amplification control. Data are means SD of three mice per group and were analyzed by one-way ANOVA with Dunnett correction for multiple comparison tests (*P < 0.05, **P < 0.01, and ***P < 0.001). All data points are the mean from technical triplicates. (C) IHC analysis of YAP and TAZ on paraffin-embedded sections of livers from 4- to 5-week-old mice. Scale bars, 50 m.

To evaluate whether YAP and TAZ are functionally active in autophagy-deficient livers, we tested for the expression of YAP/TAZ transcriptional targets in 4- to 5-week-old livers. We found that mRNA levels of connective tissue growth factor (Ctgf), amphiregulin (Areg), and cysteine-rich angiogenic inducer 61 (Cyr61), three YAP/TAZ target genes (46, 47), were all significantly up-regulated in autophagy-deficient livers (Fig. 5B and fig. S4A). At the protein level, CTGF was increased in total liver lysates of all autophagy-deficient conditions (Fig. 5A). Next, we assessed the localization of YAP and TAZ in 4- to 5-week-old autophagy-deficient livers and observed that both YAP and TAZ strongly accumulated in the ductular cells, whereas YAP and TAZ were found in the bile duct and the canal of Hering of WT counterparts (Fig. 5C and fig. S4B). Collectively, our data therefore indicate that autophagy loss in hepatocytes triggers a YAP/TAZ signature within the ductular LPC population.

YAP is turned over not only by the proteasome (48, 49), but also by autophagy as shown in recent reports (20, 50). As TAZ is a YAP homolog, we next wondered whether TAZ accumulation and activation in our autophagy-deficient livers were due to blockage of autophagy-mediated degradation of TAZ. To test more directly whether TAZ is degraded by autophagy, we first deleted ATG7 or ATG5 expression in the liver cancer cell lines HLE and Huh7 using the CRISPR-Cas9mediated gene disruption system. Next, we treated each cell line with Earles balanced salt solution (EBSS), to induce starvation-mediated autophagy, in combination with or without 200 nM bafilomycin A1 (Baf) for 2 hours to prevent lysosomal degradation of autophagosomes. We checked for the efficient disruption of ATG7 or ATG5 expression following lenti-CRISPR infection in HLE (fig. S5A) and Huh7 (fig. S5B), and we analyzed the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3)I (diffuse form in the cytosol) into LC3-II (lipidated form attached to autophagosomes), to confirm loss of autophagy. Examination of TAZ revealed that its levels did not change upon starvation-induced autophagy (EBSS), blockage of lysosomal autophagy degradation [Dulbeccos modified Eagles medium (DMEM) + Baf and EBSS + Baf], or disruption of ATG7/ATG5 (ATG7CRISPR/ATG5CRISPR) in HLE and Huh7 cells (fig. S5, A and B). Unexpectedly, we also observed that not only YAP levels accumulated under EBSS only and EBSS and Baf conditions but also this occurred in ATG7CRISPR/ATG5CRISPR cells, indicating that this was an autophagy-independent effect. Together, our data indicate that TAZ and YAP are not directly turned over by autophagy in liver cells and that the accumulation of YAP and TAZ in autophagy-deficient livers is not the result of the inhibition of the autophagy degradation pathway but instead is due to the expansion of ductular cells in vivo, which are known to express YAP and TAZ (Fig. 5 and fig. S4) (51).

Deletion of YAP partially rescued hepatomegaly, fibrosis, and tumorigenesis induced by autophagy blockage in the liver (20). As a YAP homolog, TAZ can compensate YAP activity if the latter is lost (52). Since we observed in our model that YAP and TAZ are activated within the ductular LPC population, we hypothesized that deleting both YAP and TAZ might prevent the early ductular reaction and subsequent HCC formation in autophagy-deficient livers. First, we evaluated whether TAZ has a role in the phenotype of autophagy-deficient livers. To test this, we crossed Wwtr1flox/flox (encoding TAZ) mice (53) with our liver-specific autophagy-deficient model, and we observed that loss of TAZ significantly reduced liver size of 4- to 5-week-old autophagy-deficient livers (Fig. 6A and fig. S6A). Next, we found that TAZ loss also significantly reduced the accumulation of activated -SMA+ hepatic stellate cells and collagen deposition in 4- to 5-week-old autophagy-deficient livers (Fig. 6B and fig. S6B), indicating that TAZ contributes to hepatic stellate cell activation and fibrosis in our model. In addition, TAZ loss significantly decreased SOX9+, panCK+, and EpCAM+ cells in 4- to 5-week-old autophagy-deficient livers (Fig. 6B and fig. S6B), highlighting that TAZ loss hinders the formation of ductular LPCs upon autophagy deficiency in the liver. We next compared tumor formation between Alb-Cre+; Atg7fl/fl; Pten+/fl or Alb-Cre+; Atg5fl/fl; Pten+/fl and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl or Alb-Cre+; Atg5fl/fl; Pten+/fl; Tazfl/fl in 140-day-old livers and noted that TAZ deletion caused a highly significant decrease in tumorigenesis in autophagy-deficient livers (Fig. 6, C and D, and fig. S6, C and D) that was accompanied by a significant increase in the survival of autophagy-deficient mice (Fig. 6E and fig. S6E). Last, we evaluated whether TAZ has a role in the proliferation of ductular LPCs. We found that TAZ loss did not impair the number of Ki-67+ proliferative LPCs in 4- to 5-week-old autophagy-deficient livers (fig. S7).

(A) Liver-to-body weight ratio in 4- to 5-week-old mice. Data are means SD of three mice per group and were analyzed by unpaired two tailed t test (**P < 0.01). (B) IHC analysis of hepatic stellate cell activation (-SMA), collagen deposition (Sirius Red), duct markers (SOX9 and panCK), and liver stem cell marker EpCAM on paraffin-embedded sections of livers from 4- to 5-week-old mice. Scale bars, 50 m. Left: Representative staining. Right: Quantifications. Data are mean SD of three mice per group and were analyzed by unpaired two-tailed t test (*P < 0.05, **P < 0.01, and ***P < 0.001). All data points are the mean from five pictures per mouse. (C) Macroscopic pictures of Alb-Cre+; Atg7fl/fl; Pten+/fl (top) and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl (Alb-Cre+; 7fl/fl; P+/fl; T/) (bottom) liver in 140-day-old mice. (D) Quantification of tumor numbers in Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl at 140 days. Data are means SD of five mice per group and were analyzed by unpaired two-tailed t test (***P < 0.001). (E) Kaplan-Meier analysis comparing overall survival between Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl mice (n = 5 males and n = 5 females per group). Data were analyzed by log-rank Mantel-Cox test (****P < 0.0001).

To evaluate whether there was any redundancy between YAP and TAZ in our model, we crossed Yap1flox/flox mice (53) to our liver-specific (Alb-Cre) autophagy- and TAZ-deficient model to evaluate the effect of YAP/TAZ double knockout on the ductular reaction and tumorigenesis of autophagy-deficient livers. Unexpectedly, we observed that 40% (9 of 22 mice) of YAP-deficient mice developed jaundice within 6 to 8 weeks regardless of Atg7, Atg5, Pten, or Wwtr1 genotype. This is likely because YAP is highly expressed in the bile duct of WT mice (Fig. 5C and fig. S4B), and the Albumin promoter driving Cre recombinase expression is expressed in hepatoblasts, the embryonic progenitor cells generating hepatocytes and cholangiocytes (54). YAP deletion in our Albumin-Cre model can therefore impair cholangiocyte function in the bile duct leading to acute jaundice. To overcome this phenotype for long term studies, we used AAV8-TBG-Cre adenovirus to induce Cre recombination more specifically in the hepatocytes of our Atg7flox/flox; Ptenflox/flox; Yap flox/flox; Tazflox/flox model (Fig. 7A). First, we assessed the effect of YAP/TAZ deletion on the hepatomegaly and ductular reaction of autophagy-deficient livers 3 weeks following AAV8-TBG-Cre recombination and confirmed the recombination of Atg7, Pten, Yap, and Wwtr1 alleles in AAV8-TBG-Creinfected livers (fig. S8). We found that although YAP or TAZ deletion significantly reduced hepatomegaly of autophagy-deficient livers (Fig. 7B), YAP/TAZ double knockout mice significantly restored liver size to that observed in nonrecombined counterparts infected with the AAV8-TBG-null adenovirus (Fig. 7B). In addition, we noted that while the individual deletion of Yap or Taz significantly impaired the formation of SOX9+ cells in autophagy-deficient livers (Fig. 7, C and D), only YAP/TAZ codeletion completely blocked the formation of SOX9+ cells in autophagy-deficient livers (Fig. 7, C and D). In this AAV8-TBG-Cre model, Atg7/; Pten/ mice had to be culled because of hepatomegaly and did not develop tumors at humane end point. To evaluate the role of YAP/TAZ loss in the tumorigenesis of autophagy-deficient livers, we infected Atg7flox/flox; Pten+/flox; Yapflox/flox; Tazflox/flox with AAV8-TBG-Cre adenovirus and assessed tumor formation 140 days following AAV8 infection (Fig. 7E). We observed that while Yap or Taz deletion significantly impaired tumorigenesis in autophagy-deficient livers (Fig. 7, F and G), only YAP/TAZ codeletion completely prevented tumor formation (Fig. 7, F and G). Our data therefore show that deleting YAP and TAZ suppresses the ductular reaction and tumorigenesis of autophagy-deficient livers. However, in this context, we observed functional redundancy between YAP and TAZ, and only the combined deletion of both these genes could revert the effects on tissue overgrowth and tumor development.

(A) Schematic representation. Eight-week old Atg7fl/fl; Ptenfl/fl Yapfl/fl (Yfl/fl) and/or Tazfl/fl (Tfl/fl) mice were infected with AAV8-TBG-Cre and aged for 3 weeks before hepatomegaly and ductular reaction analysis. (B) Liver-to-body weight ratio in mice 3 weeks after AAV8 infection. Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, ***P < 0.001, and ****P < 0.0001). (C) IHC analysis of the duct marker SOX9 on paraffin-embedded sections of livers from mice 3 weeks after AAV8 infection. Scale bars, 50 m. (D) Quantification of SOX9 from (C). Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (****P < 0.0001). All data points are the mean from five pictures per mouse. (E) Schematic representation. Eight-week-old Atg7fl/fl; Pten+/fl; Yapfl/fl and/or Tazfl/fl mice were infected with AAV8-TBG-Cre and aged for 140 days before tumor analysis. (F) Macroscopic pictures from 140 days after AAV8-Cre livers. (G) Quantification of tumor numbers in 140 days after AAV8-Cre livers. Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, **P < 0.01, and ****P < 0.0001). All data points are the mean from five pictures per mouse. Xfl/fl, AAV8-null infected; X/, AAV8-Cre infected.

We report a new model for extensive ductular reaction upon deletion of ATG5 or ATG7 and PTEN in the murine liver. Although Pten-deficient livers develop steatosis and HCC (37), we observed that hepatic Pten deletion alone did not initiate liver damage, inflammation, hepatic stellate cell activation, fibrosis, or a ductular reaction in young livers, but these effects were observed on hepatic deletion of ATG5 or ATG7. ATG5 and ATG7 are two proteins that are essential for the stage of autophagy that involves LC3 conjugation. ATG5 and ATG7 are also important for two other processes that involve the LC3 conjugation machinery: LC3-associated phagocytosis (LAP) (55) and LC3-associated endocytosis (LANDO) (56). We consider, however, that the core observations in our study relating to tumor development and liver injury are connected to autophagy, as previous studies have shown that they can be reversed by concomitant deletion of the autophagy adapter protein p62 (11, 22, 57), and autophagy adapter proteins are not thought to be involved in LAP or LANDO (58). We cannot fully discount that some of the effects we observe on deletion of ATG5 or ATG7 may be related to LAP or LANDO rather than autophagy or a combination thereof. Future studies to clarify this point using deletion of other factors such as FIP200 or ATG13 that are involved in autophagy, but not LAP and LANDO (5962), would certainly be merited to investigate this possibility.

Autophagy is impaired in Pten-deficient mice due to mTORC1 activation; however, autophagy is not blocked in Pten-deficient livers (63). LC3 is still conjugated to phosphatidylethanolamine leading to autophagosome and autolysosome formation when Pten expression is lost (63). This dictates an important role for autophagy in hepatocytes to prevent the microenvironmental remodeling and ductular reaction in healthy livers, with Pten cooperating with the autophagy-specific phenotype. Pten loss induces cellular senescence to protect from tumorigenesis in different models (64, 65). However, we noticed the presence of apoptotic hepatocytes following autophagy abrogation and Pten deletion. The extent of injury in hepatocytes determines their fate toward senescence or cancer (66). Acute injury in hepatocytes results in senescence (67), while chronic injury does not activate senescence in hepatocytes, ultimately leading to HCC (66). Autophagy degrades damaged mitochondria, a process named mitophagy, to maintain cellular homeostasis. In hepatocytes, loss of autophagy leads to ROS accumulation, damaged mitochondria, and dysfunction (11, 22, 68, 69). We suggest that the persistence of chronic damage and defects in damaged mitochondria clearance by mitophagy drive apoptosis and tumorigenesis in our autophagy- and Pten-deficient livers.

In our autophagy- and Pten-deficient model, we observed that following liver injury, hepatocytes dedifferentiate into ductular LPCs. This ductular reactive phenotype is not unique to the loss of autophagy as it has previously been observed in animal models subjected to diet modification, e.g., a diet enriched in 3,5-diethoxycarboncyl-1,4-dihydrocollidine (70) or choline-deficient, ethionine-supplemented diet (71). This indicates that the ductular reaction is likely to be a secondary effect of autophagy inhibition due to liver damage caused by autophagy loss. The origin of the ductular reaction in rodents is still controversial, with reports indicating the role of biliary cells (28, 29) or hepatocytes (30, 31) in forming LPCs with the capacity for generating new hepatocytes upon liver injury. Here, we show in a genetically modified mouse model that ductular reactive cells arise from mature hepatocytes upon injury induced by autophagy deficiency. The cellular plasticity of human hepatocytes can also generate ductular cells in a transplantation mouse model (30), strengthening the hepatocyte origin of the ductular reaction in human liver diseases.

The plastic differentiation program of the ductular reaction for liver regeneration is defined by the origin of the injuries. Following bile duct injury, resident LPCs/biliary cells (26) and hepatocyte-derived LPCs (72) regenerate biliary cells. When hepatocyte function is impaired, resident LPCs/biliary cells (28, 29, 73) or hepatocyte-derived LPCs (30, 31, 74) generate new hepatocytes. The decision to recruit biliary cells or hepatocytes during the ductular reaction remains elusive, and future studies will be required to shed further light on this mechanism.

Autophagy loss has been previously shown to give rise to HCC in mice (20). Our results suggest that the hepatocyte-derived ductular reaction gives rise to HCC in autophagy-deficient livers. While some studies conclude that the ductular reaction is not involved in liver carcinogenesis (34, 35, 74), other studies do report a role for the ductular reaction in initiating HCCs (32, 33). Although all these studies recombine LPCs for lineage tracing, they differ with respect to the timing between the induction of LPC labeling and the start of the injury. Recombination of LPCs for lineage tracing before inducing liver injury (34, 35, 74) does not label hepatocyte-derived LPCs, excluding them from the lineage tracing of HCCs. In contrast, recombination of LPCs for lineage tracing following liver injury results in LPC-derived HCCs (32, 33). In our autophagy- and Pten-deficient model, we report that hepatocyte-derived LPCs generate SOX9+ hepatocytes that give rise to HCC. The ability of LPCs to induce tumorigenesis has been controversial since it is generally accepted that HCC originates from hepatocytes. Here, we reconcile these findings by showing that HCC does originate from hepatocytes, but these hepatocytes, early upon liver injury, dedifferentiate into LPCs to attempt to regenerate liver function, before transforming into HCC.

In human liver diseases, the accumulation of LPCs is observed in nonalcoholic steatohepatitisinduced cirrhosis preceding HCC (75), and the presence of peritumoral ductular reaction is a poor prognostic factor for human HCC after resection (76), indicating the importance of targeting the ductular reaction in human liver diseases. The gene signature of autophagy-deficient mice is similar to the human transcriptomes of nonalcoholic fatty livers (20), and rat livers from rats fed a high-fat diet reduce their autophagy function (77). Restoring autophagy could therefore be a beneficial treatment in injured livers harboring a ductular reaction.

Mechanistically, we report that YAP and TAZ cooperate to drive hepatocyte dedifferentiation and tumorigenesis in autophagy-deficient livers. Unlike a previous study on YAP (20), we uncovered that TAZ also plays a role in promoting hepatomegaly, ductular reaction, stromal activation, fibrosis, and tumorigenesis in autophagy-deficient livers. TAZ deletion alone, similar to YAP deletion alone (20), only impaired carcinogenesis in autophagy-deficient livers. However, TAZ loss did not impair the proliferative outgrowth of the ductular LPC population. Here, we speculate that TAZ is involved in the differentiation switch in our model as its homolog YAP can directly drive hepatocyte dedifferentiation (31), and, more recently, YAP/TAZ have been described as regulators of stemness and cell plasticity in glioblastoma (78). We found that YAP and TAZ are not directly turned over by autophagy and that their accumulation in the absence of autophagy in vivo is associated with the increased presence of ductular cells, which are known to express YAP and TAZ (79). YAP and TAZ are mechanosensors and mechanotransducers (80), and their activation is linked to the stiffness of the extracellular matrix (81). As we noted a significant increase in extracellular matrix remodeling and fibrosis (Fig. 1E and fig. S1, F and G) correlating with a significant increase in YAP+/TAZ+ ductular LPCs in our models, we suggest that YAP and TAZ are also activated in response to the microenvironment changes following autophagy and PTEN deletion in the liver. Building on these findings, we observed that only the combined deletion of YAP and TAZ prevented the emergence of hepatocyte-derived LPCs that initiate tumorigenesis in autophagy-deficient livers. Our study uncovered a role for autophagy in suppressing the emergence of hepatocyte-derived ductular LPCs that can give rise to HCCs via concomitant activation of YAP and TAZ.

Male and female animals were housed in a pathogen-free environment and kept under standard conditions with a 12-hour day/night cycle and access to food and water ad libitum. All in vivo experiments were carried out under guidelines approved by the Glasgow University Animal Welfare and Ethical Review Body and in accordance with U.K. Home Office guidelines under license P54E3DD25. As described previously (82), Alb-Cre+ mice [RRID (research resource identifier): MGI:2176228] were crossed to Atg7fl/fl (68) (RRID: MGI:3590136) or Atg5fl/fl (83) (RRID: MGI:3612279) and Ptenfl/fl (84) (RRID: MGI:2182005) to generate the different combinations on a mixed background. Subsequently, Atg7fl/fl; Ptenfl/fl and Atg5fl/fl; Ptenfl/fl mice were crossed to Yap1fl/fl; Wwtr1fl/fl (the Jackson laboratory, stock 030532, RRID: IMSR_JAX:030532) (53) animals to generate all the different combinations. Experimental cohort (males and females) sizes were based on previous similar studies that have given statistically significant results while also respecting the limited use of animals in line with the 3R system: replacement, reduction, and refinement. All treatment studies were randomized but did not involve blinding. Genotyping was performed by Transnetyx. To lineage trace the ductular cell origin, we crossed our model with the Rosa26-mtdTomato-mEGFP mouse (the Jackson laboratory, stock 007576, RRID: IMSR_JAX:007576) (85).

In AAV8 studies, AAV8 recombination was performed as previously described (67). Briefly, viral particles [2 1011 genomic copies per mouse] of AAV8.TBG.PI.Cre.rBG (Addgene, catalog no. 107787-AAV8), AAV8.TBG.PI.eGFP.WPRE.bGH (Addgene, catalog no. 105535-AAV8), or AAV8.TBG.PI.Null.bGH (Addgene, catalog no. 105536-AAV8) were injected in 6-week-old (AAV8-GFP and AAV8-null) or 8-week-old (AAV8-Cre and AVV8-null) mice via tail vein in 100 L of phosphate-buffered saline (PBS).

Mice were euthanized by CO2 inhalation followed by cervical dislocation, and blood was harvested by cardiac puncture in accordance with U.K. Home Office guidelines. Tissues were weighed and stored immediately at 80C or in paraffin blocks after fixation in 10% formalin (in PBS) for 24 hours, followed by dehydration in 70% ethanol before embedding. Blood samples (EDTA-plasma and serum) were stored at 80C following 10-min centrifugation at 900g at 4C. Serum was sent to the Veterinary Diagnostic Services (University of Glasgow) for ALT, AST, ALP, and GGT analyses.

Plasma AFP levels were assessed using the enzyme-linked immunosorbent assay (ELISA) kit (catalog no. ab210969) according to the manufacturers instruction. Each sample was analyzed in triplicate.

For immunohistochemical (IHC) or immunofluorescence (IF) studies, paraffin-embedded sections were deparaffinized, rehydrated, and heated to 95 to 97C either in Lab Vision Citrate Buffer for heat-induced epitope retrieval (pH 6.0) (Thermo Fisher Scientific, catalog no. 12638286), EnVision FLEX Target Retrieval Solution, High pH (Agilent, catalog no. K8004), BOND Epitope Retrieval Solution 2 (ER2) (Leica, catalog no. AR9640), or Antigen Unmasking Solution, Citric Acid Based (Vector Laboratories, catalog no. H-3300) for antigen retrieval, depending on the primary antibody used. Primary antibodies used for IHC analyses: Ly6G (Bio X Cell, catalog no. BE0075-1, RRID: AB_1107721, rat, ER2; 1:60,000), -SMA (Sigma-Aldrich, catalog no. A2547, RRID: AB_476701, mouse, citric acid; 1:25,000), CC3 (Asp175, Cell Signaling Technology, catalog no. 9661, RRID: AB_2341188, rabbit, ER2; 1:500), SOX9 (Millipore, catalog no. AB5535, RRID: AB_2239761, rabbit, high pH; 1:500), CK19 (Novus, catalog no. NB100-687, RRID: AB_2265512, rabbit, high pH; 1:100), panCK (Lab Vision, catalog no. MS-343-P, RRID: AB_61531, mouse, Citric acid; 1:100), EpCAM (Abcam, catalog no. ab71916, RRID: AB_1603782, rabbit, high pH; 1:1500), CD133 (Abcam, catalog no. ab19898, RRID: AB_470302, rabbit, citrate pH 6; 1:200), CD44 (BD Biosciences, catalog no. 550538, RRID: AB_393732, rat, ER2; 1:300), GFP (Cell Signaling Technology, catalog no. 2555, RRID: AB_10692764, rabbit, ER2; 1:600), red fluorescent protein (Rockland, catalog no. 600-401-379, RRID: AB_2209751, rabbit, high pH; 1:1000), YAP (Cell Signaling Technology, catalog no. 4912, RRID: AB_2218911, rabbit, high pH; 1:50), WW domain containing transcription regulator 1 (WWTR1)/TAZ (Sigma-Aldrich, catalog no. HPA007415, RRID: AB_1080602, rabbit, high pH; 1:100), and Ki-67 (Cell Signaling Technology, catalog no. 12202, RRID: AB_2620142, rabbit, ER2; 1:1000). Primary antibodies were incubated with sections for 40 min at room temperature or overnight at 4C. For IHC analysis, primary antibodies were detected using mouse or rabbit EnVision+ System kits (Agilent, catalog no. K4001 and K4006) or ImmPRESS horseradish peroxidase (HRP) goat anti-rat immunoglobulin G (IgG) polymer detection kit (Vector Laboratories, catalog no. MP-7404) and 3,3-diaminobenzidine substrate (Agilent, catalog no. K4011). Slides were then counterstained with hematoxylin solution. Images were obtained on a Zeiss AX10 (light microscopy) at a 20 or 40 magnification.

For IF analysis, SOX9/GFP immunofluorescent primary antibodies were applied sequentially. First, slides were incubated with a chicken polyclonal GFP antibody (Abcam, catalog no. ab13970, RRID: AB_300798, citrate; 1:200) overnight at 4C and was detected using a biotinylated goat anti-chicken (Vector Laboratories, catalog no. BA-9010, RRID: AB_2336114; 1:200) coupled to Avidin-HRP (Vector Laboratories, PK-7100) and a PerkinElmer TSA Plus Cyanine 3 signaling amplification kit (NEL744B001KT; 1:50). This was followed by a second antigen retrieval to denature any antibodies in the tissue. Slides were then incubated with a rabbit monoclonal SOX9 antibody (Abcam, catalog no. ab185230, RRID: AB_2715497, citrate; 1:500) overnight at 4C and detected using a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, catalog no. A-21206, RRID: AB_2535792; 1:200). Slides were then counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were obtained on a Zeiss 710 confocal microscope at a 20 magnification. For collagen staining, sections were rehydrated and then immersed in Picro Sirius Red solution [0.1% Direct Red 80 (Sigma-Aldrich, 41496LH) and 0.1% Fast Green FCF (Raymond Lamb, S142-2) diluted in aqueous picric acid solution] for 2 hours.

HLE and Huh7 were grown in DMEM (Gibco, 21969-035) supplemented by 10% fetal bovine serum (FBS; Gibco, 10270-106), 2 mM glutamine (Gibco, 25030-032), streptomycin (100 g/ml), and penicillin (100 U/ml; Gibco, 15140-122) (complete DMEM) at 37C and 5% CO2. For starvation-induced autophagy experiments, cells were washed twice in PBS and starved in EBSS (Sigma-Aldrich, E2888) containing or not 200 nM Baf (LC Labs, B-1080) for 2 hours. HLE and Huh7 cell lines were provided by T. Bird.

Lentiviruses were produced using human embryonic kidney (HEK) 293T cells using calcium/phosphate transfection protocol. Cells were transfected overnight with lentiviral, packaging, and envelope plasmids (pPAX2 and pVSVG). The following day, media were replaced by complete DMEM containing 20% FBS for 24 hours. Then, virus-enriched media were collected, filtered (0.45 m), supplemented with polyprene (4 g/ml; Sigma-Aldrich, H9268), and transferred to recipient cells. In the meantime, HEK293T cells were kept in DMEM containing 20% FBS for an additional 24 hours to perform a second round of infection of recipient cells as described before. Last, infected cells were selected with puromycin (2 g/ml; Sigma-Aldrich, P9620) for 10 days. The following single-guide RNA sequences were used in this study: human ATG7, 5-GAA GCT GAA CGA GTA TCG GC-3 (86); human ATG5, 5-AAG AGT AAG TTA TTT GAC GT-3 (86); nontargeting control, 5-GTA GCG AAC GTG TCC GGC GT-3 (87).

Livers were dissociated using a Precellys Evolution (Bertin Technologies) and lysed in 1% Triton X-100, 0.1% SDS, 50 mM Hepes (pH 7.5), 150 mM NaCl, 100 mM NaF, and 10 mM EDTA, supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog no. 87786). After 15-min centrifugation at 12,000g at 4C, the supernatant was removed, and the concentration of solubilized proteins was determined with the Pierce bicinchoninic acid assay (Thermo Fisher Scientific, catalog no. 23225). Protein lysates were separated by SDSpolyacrylamide gel electrophoresis with Criterion TGX Stain-Free precast gels (Bio-Rad) or the NuPAGE 4 to 12% bis-tris gel (Invitrogen) and blotted onto polyvinylidene difluoride membranes (Merck). Criterion TGX Stain-Free precast gels (Bio-Rad) were activated using the ChemiDoc (Bio-Rad) to detect total protein levels. Total protein level was measured before and after transfer. Western blot analysis was performed according to the manufacturers instructions for Criterion TGX Stain-Free precast gels or for the NuPAGE 4 to 12% bis-tris gel (Invitrogen). The following antibodies were used at a dilution of 1:1000 unless otherwise stated: p-YAP (Cell Signaling Technology, catalog no. 13008, RRID: AB_2650553), YAP (Cell Signaling Technology, catalog no. 4912, RRID: AB_2218911; 1:750), p-TAZ (Cell Signaling Technology, catalog no. 59971, RRID: AB_2799578), YAP/TAZ (Cell Signaling Technology, catalog no. 8418, RRID: AB_10950494), CTGF (Abcam, catalog no. ab125943, RRID: AB_2858254), ATG7 (Cell Signaling Technology, catalog no. 8558, RRID: AB_10831194), PTEN (Cell Signaling Technology, catalog no. 9559, RRID: AB_390810), extracellular signalregulated kinase 2 (ERK2; Santa Cruz Biotechnology, catalog no. sc-154, RRID: AB_2141292), LC3B (Cell Signaling Technology, catalog no. 2775, RRID: AB_915950), ATG5 (Cell Signaling Technology, catalog no. 12994, RRID: AB_2630393), glyceraldehyde-3-phosphate dehydrogenase (Abcam, catalog no. ab9485, RRID: AB_307275), anti-rabbit IgG HRP-linked (Cell Signaling Technology, catalog no. 7074, RRID: AB_2099233; 1:4000), and anti-mouse IgG HRP-linked (Cell Signaling Technology, catalog no. 7076, RRID: AB_330924; 1:4000).

RNAs were extracted from livers using the RNeasy Mini Kit (QIAGEN, catalog no. 74101) and quantified using a NanoDrop200c (Thermo Fisher Scientific). Complementary DNAs (cDNAs) were produced using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, catalog no. 4388950) according to the manufacturers instruction. Quantitative polymerase chain reactions (qPCRs) were performed using the DyNAmo HS SYBR Green qPCR Kit (Thermo Fisher Scientific, catalog no. F-410) on a Step-One Plus (Applied Biosystems) as follows: 20 s at 95C, followed by 40 cycles of 3 s at 95C, and 30 s at 60C. mRNA quantification was calculated using Ct method. The following mouse primers were used: mouse Ctgf (QIAGEN, QT00174020), mouse Ctgf (QIAGEN, QT00096131), mouse Cyr61 (QIAGEN, QT00245217), mouse Areg (QIAGEN, QT00112217), 18S forward (5-GTAACCCGTTGAACCCCATT-3), and 18S reverse (5-CCATCCAATCGGTAGTAGCG-3).

For IHC studies, five representative pictures were taken per mouse and were analyzed using Fiji software. For all in vivo studies, data are shown as means SD. Sample normality was assessed by Shapiro-Wilk test. Statistical significances were determined by two-tailed unpaired Students t test for two-group comparison, two-way analysis of variance (ANOVA) with Tukey or Dunnett for multiple group comparison, and log-rank (Mantel-Cox) test for survival comparison using GraphPad Prism software. Results were considered statistically different when *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 with ns indicating no significance.

J. OPrey, J. Sakamaki, A. D. Baudot, M. New, T. Van Acker, S. A. Tooze, J. S. Long, K. M. Ryan, in Methods in Enzymology, vol. 588 of Molecular Characterization of Autophagic Responses, Part B, L. Galluzzi, J. M. Bravo-San Pedro, G. Kroemer, Eds. (Academic Press, 2017), pp. 79108.

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Autophagy suppresses the formation of hepatocyte-derived cancer-initiating ductular progenitor cells in the liver - Science Advances

Brave Nathaniel Nabena smiles from his hospital bed moments before a life-saving procedure.

The nine-year-old had a vital stem cell transplant at Great Ormond Street Hospital on Wednesday after Sunday People readers helped raised more than 215,000.

Nathaniel, battling acute myeloid leukaemia, was on a drip for 30 minutes as umbilical cord stem cells were fed into his body.

Afterwards, dad Ebi said: Nathaniel is very happy. It was amazing to finally get to this point we have all been waiting for.

The youngster was admitted a fortnight ago and had five doses of chemo over ten days to prepare him for the procedure.

How brave has Nathaniel been? Have your say in comments below

Mum Modupe, 38, was able to spend time with him before his transplant.

Consultants warn he faces weeks of sickness as his body reacts to the new cells with symptoms including vomiting and a fever.

Ebi, 45, said: His doctors hope to see improvements after five weeks. It is so hard to see him so exhausted but I dont have a choice. We are grateful to have this done. Our fingers are crossed to see what happens.

For now, Nathaniel has a compromised immune system and is susceptible to falling ill, so he will be staying on the ward.

Stars including Simon Cowell, David Walliams, Katie Price and JLS singer Aston Merrygold rallied to support him after we told of the desperate race to fund treatment.

Nathaniels left eye was removed in his home country of Nigeria a year ago, due to myeloid sarcoma cancer. He was diagnosed with AML in the UK in November after coming here to have a prosthetic eye fitted.

Nathaniel was told a stem-cell transplant was his only hope for survival but it would cost 201,000 as he is not a British citizen. Ebi and Modupe were initially told it could cost as much as 825,000 but the figure was revised after doctors waived their fees and offered to treat him in their own time.

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The lad was admitted to GOSH on May 24 after generous Brits rushed to help the family raise cash.

Business analyst Ebi, who is staying at the hospitals family quarters, said: Ive been there the whole time. When he is not sleeping he is passing the time playing his games.

We sometimes talk about when he gets better and how exciting that will be. This is a difficult thing for him to go through, but Nathaniel is being brave, he is well in himself.

In acute myeloid leukaemia, unhealthy blood-forming stem cells grow quickly in the bone marrow.

This prevents it from making normal red blood cells, white blood cells and platelets meaning the body cannot fight infections or stop bleeding.

A stem cell transplant, also known as a bone marrow transplant, can help AML patients stimulate new bone marrow growth and restore the immune system.

Before treatment, patients need high doses of chemo and sometimes radiotherapy.

This destroys existing cancer and bone marrow cells and stops the immune system working, to cut the risk of transplant rejection.

In an allogeneic transplant, stem cells are taken from a family member, unrelated donor or umbilical cord blood. In Nathaniels case, it was from a cord.

They are then passed into the patients body through a line inserted in a large, central vein, in a process that takes up to two hours.

You can also remove stem cells from the patients body and transplant them later, after any damaged or diseased cells have been removed this is called an autologous transplant.

The survival rate after a transplant for patients with acute leukaemia in remission and using related donors is 55% to 68%, according to Medicine Net. If the donor is unrelated, it is 26% to 50%.

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Brave Nathaniel Nabena, 9, all smiles as he has life-saving procedure - thanks to you - The Mirror

Venetoclax (Venclexta) plus fulvestrant (Faslodex) did not lead to better outcomes compared to fulvestrant alone in patients who had locally advanced or metastatic estrogen receptor (ER)-positive, HER2-negative breast cancer that was previously treated with a CDK4/6 inhibitor, according to findings from the phase 2 VERONICA trial (NCT03584009), which was presented during the 2021 ASCO Annual Meeting.

At a median follow-up of 9.9 months, the clinical benefit rate (CBR)was 11.8% (95% CI, 4.44%-23.87%) with venetoclax/fulvestrant vs 13.7% (95% CI, 5.7%-26.26%) with fulvestrant alone, translating to a risk difference of -1.96%(95% CI, -16.86%-12.94%).

The primary analysisof VERONICA revealed a largely endocrine-refractory population of patients. Venetoclax added to fulvestrant did not improve CBR orprogression-free survival [PFS], [nor did]overall survival [OS]favor [the combination],lead study author Geoffrey J. Lindeman, MD,joint head of the, Stem Cells and Cancer Division at The Walter and Eliza Hall Institute of Medical Research,saidin a virtual presentation of the data.

Despite the use of the combination of aCDK4/6 inhibitorandchemotherapy, whichhas become the standard frontline therapy for patients withmetastaticER-positive, HER2-negative breast cancer, disease progressionis inevitable.

BCL-2 is a pro-survival protein that is overexpressed in the majority of primary and relapsed ER-positive breast cancers.The BCL-2 inhibitor venetoclaxhas shown promising activity in patients withendocrine-nave, ER-positive, BCL-2positive metastatic breast cancer.

To that end, investigators evaluated the activity of adding the BCL-2 inhibitor to fulvestrant in patients withprogressive ER-positive, HER2-negative disease.

Eligibility criteria stipulated that females,18 years of age or older,had to have locally advanced or metastatic ER-positive, HER2-negativebreast cancer, received 2 or fewer lines of therapy in the locally advanced or metastatic setting without chemotherapy, received a CDK4/6 inhibitorat least 8 weeks before enrollment, and have measurable disease.

Patients were randomized 1:1 to 800 mg of oral, dailyvenetoclax (n = 51) plus 500 mg of intramuscular fulvestrant on day 1 and 15 of cycle 1 and day 1 ofeach 28-day cycle thereafteror fulvestrant alone (n = 52). Treatment was continued until disease progression, unacceptable toxicity, withdrawal of consent, death, or predefined study end.

CBR,defined as thetotal complete response(CR), partial response(PR), and stable disease rate after at least 24 weeks, served as the primary end point of the study. Secondary end pointsincludedPFS, OS, objective response rate (ORR)defined as the total CR and PR rateand duration of response (DOR).

Additional end points included safety and tolerability, biomarker analysis, pharmacokinetics, and patient-reported outcomes.

The primary analysis took place on August 5, 2020, and the updated analysis took place in April 2021.

Regarding baseline demographics, the median age was 58 years in thevenetoclax arm vs 59.5 years in the fulvestrant-alone arm.Approximately half of all patients had an ECOG performance status of 0 in both arms,at 54.9% and 59.6%, respectively. Moreover, in both arms, the majority of patients were White(78.4% vs 88.5%, respectively), had ductalhistology(78.4% vs 65.4%, respectively), at least 1 visceral metastatic lesion(92.2% vs 82.7%, respectively), and 1 prior line of endocrine therapy in the metastatic setting(80.4% vs 82.7%, respectively).

All patients had received prior endocrine therapy in the venetoclax and fulvestrant-alone arms, whereas approximately half had receivedadjuvant chemotherapy (58.8% vs 51.9%, respectively), and less than a quarter had received prior neoadjuvant chemotherapy (23.5% vs 13.5%, respectively).

The median duration of exposure to prior treatment with a CDK4/6 inhibitorin the metastatic settingwas 15 months in the venetoclax arm vs 16.5 months in the fulvestrant-alone arm,with palbociclib (Ibrance; 56.9% vs 75%, respectively) and ribociclib (Kisqali; 43.1% vs 25%, respectively).

Regarding BCL-2 status, more patients had high expressionin the venetoclax and fulvestrant-alone arms (64.7% vs 65.4%, respectively) thanlow expression (35.3% vs 34.6%, respectively).

Biomarker statusin the venetoclax and fulvestrant-alone arms, respectively,indicated the presence ofmutations in the PIK3CA (39.6% vs 30.4%),ESR1 (43.8% vs 41.3%),TP53 (47.9% vs 34.8%), andRB1 (18.8% vs 8.7%)genes.

Additional results demonstrated that the ORR was3.9% in the venetoclax arm vs 5.9% in the fulvestrant-alone arm and consisted all of PRs.

The median PFS was 2.69 months (95% CI, 1.94-3.71) in the venetoclax arm vs 1.94 months(95% CI, 1.84-3.55)in the fulvestrant-alone arm (HR, 0.94; 95% CI, 0.61-1.45;P= .7853).The 6-month PFS rates were 12.3% vs 18.8%, respectively.

The OS data were not mature at the time of the primary analysis but did not favor the venetoclax arm. The median OS was 16.76 months (95% CI, 10.12-not evaluable [NE]) in the venetoclax arm vs NE (95% CI, 16-NE) in the fulvestrant-alone arm (HR, 2.56; 95% CI, 1.11-5.89;P= .0218).The updated analysis showed comparable results, with a numerically lower hazard ratio of 1.85(95% CI, 1.01-3.39).

Notably, similar CBR and PFS was observed between arms,irrespective of BCL-2 expression.

However, increased CBR and PFS was reported in thePIK3CAwild-type subgroupin an exploratory analysis. Here, the CBR was20.7% in the venetoclax arm(n = 29) vs 9.7% in the fulvestrant-alone arm(n = 31).The median PFS was 3.71 months (95% CI, 1.94-4.53)vs 1.87 (95% CI, 1.74-3.55), respectively(HR, 0.66; 95% CI, 0.38-1.17;P=.1549).

Ahigher number of deaths was reported in the venetoclax arm vs the fulvestrant-alone arm primarily because of progressive disease at least 28 days after the last dose of study treatment. A similar trend was reported in the updated analysis.

The safety profile of the combination was consistent with the known safety profile of each agent alone, and no new signals were identified.

The occurrence of at least 1 adverse effect (AE) was reported in 94% of patients in the venetoclax arm vs 76.5% of patients in the fulvestrant-alone arm. Grade 3 or 4 AEs were reported in 26% vs 11.8% of patients, respectively.Serious AEs occurred in 8% vs 2% of patients, respectively.One case of urosepsis leading to death occurred in the venetoclax arm but was unrelated to the study drug.

Treatment-related AEs leading to drug withdrawal occurred in 8% of patients in the venetoclax arm vs 0% of patients in the fulvestrant-alone arm.AEs leading to dose modification or interruption occurred in 44% vs 2% of patients, respectively.

The most common grade 3 or 4 AEs in the venetoclax arm included fatigue (6%), neutropenia (12%), lymphopenia (4%), and dyspnea (4%)vsa 2% incidence of grade 3 or 4 fatigue in the fulvestrant-alone arm.

It remains unclear whether a BCL-2 inhibitor would be effective in an endocrine therapyresponsive, CDK4/6 inhibitornave setting, concluded Lindeman.

Reference

Lindeman GJ,Bowen R, Jerzak KJ,et al. Results from VERONICA: a randomized, phase II study of second-/third-linevenetoclax + fulvestrant vs. fulvestrantalone in estrogen receptor-positive, HER2-negative, locally advanced, or metastatic breast cancer.J Clin Oncol. 2021;39(suppl 15):1004.doi:10.1200/JCO.2021.39.15_suppl.1004

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Venetoclax Plus Fulvestrant Did Not Improve Outcomes Compared to Fulvestrant Alone in ER+/HER2- Breast Cancer - Cancer Network

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Magenta Therapeutics (Nasdaq: MGTA), a clinical-stage biotechnology company developing novel medicines to bring the curative power of stem cell transplants to more patients, today announced additional positive results from a Phase 2 clinical trial of MGTA-145 and plerixafor in patients with multiple myeloma at the American Society of Clinical Oncology (ASCO) Annual Meeting, being held virtually June 4-8, 2021.

We are very pleased to see continued favorable results for MGTA-145 and plerixafor for stem cell mobilization and collection in patients with multiple myeloma, said Jason Gardner, D.Phil., President and Chief Executive Officer, Magenta Therapeutics. These results build on those previously disclosed from this study and the Phase 1 trials to further demonstrate MGTA-145 and plerixafors potential as a rapid, reliable, well-tolerated approach to stem cell mobilization and collection, which has positive implications for patients and donors.

Additional Results MGTA-145 Multiple Myeloma Phase 2 Clinical Trial

The investigator-initiated, 25-patient Phase 2 clinical trial is designed to evaluate the ability of MGTA-145, in combination with plerixafor, to mobilize and collect hematopoietic stem cells for autologous stem cell transplant in patients with multiple myeloma. This study is led by Surbhi Sidana, M.D., Assistant Professor of Medicine in the Division of Blood and Marrow Transplantation and Cellular Therapy at Stanford University School of Medicine.

Previously reported results from this trial were announced on May 12, 2021, in a published abstract for the European Hematology Association (EHA) Congress and provided preliminary data from the initial cohort of 10 patients.

Summary of cumulative results through data cut-off date:

As indicated previously, this trial has broad and clinically representative inclusion criteria and includes patients that represent the general transplant-eligible population of patients with multiple myeloma. Patients enrolled in this trial included those patients with risk factors that could impact stem cell mobilization and collection, such as myeloma-directed therapies that are known to impact stem cell collection, previous malignancy treated with chemotherapy and/or radiation, and other co-morbid conditions. Mobilization agents may be less effective in patients with multiple risk factors. Final clinical data from this trial are anticipated by the end of 2021. MGTA-145 is also being evaluated for its ability to mobilize and collect stem cells from donors for allogenic transplant in patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in a Phase 2 trial, and an additional Phase 2 study is planned to initiate in patients with sickle cell disease in the second half of 2021.

ASCO Poster Presentation

Title: Phase 2 Study of MGTA-145 + Plerixafor for Rapid and Reliable Hematopoietic Stem Cell (HSC) Mobilization for Autologous Stem Cell Transplant in Multiple Myeloma (Abstract #8023)

Author: Surbhi Sidana, M.D., Assistant Professor of Medicine in the Division of Blood and Marrow Transplantation and Cellular Therapy, Stanford University School of MedicinePoster Session: Hematologic Malignancies Plasma Cell DyscrasiaDate/Time: All e-posters are now available in the ASCO Annual Meeting virtual platform

These results will also be presented as an encore at the EHA Virtual Congress, available via the conferences virtual platform on Friday, June 11 at 3:00am EDT / 9:00am CEST.

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Magenta Therapeutics (MGTA) Announces Additional Preliminary Positive Results from Ongoing Phase 2 Clinical Trial of MGTA-145 and Plerixafor in...

PROVIDENCE, R.I.[Brown University] New research provides insights into the treatment of Christianson syndrome (CS), an X-linked genetic disease characterized by reduced brain growth after birth, intellectual disability, epilepsy and difficulties with balance and speech.

One of the major challenges in developing treatments for human brain disorders, like CS, is developing an experimental system for testing potential therapeutics on human neurons, said study senior author Dr. Eric Morrow, an associate professor of molecular biology, neuroscience and psychiatry at Brown University. In recent years, advanced stem cell therapies that use tissues from patients have provided powerful new approaches for engineering human neurons from the patients themselves, who may undergo the treatment in the future.

For the study, published in Science Translational Medicine on Feb. 10, 2021, Morrow and his colleagues obtained blood samples from five CS patients and the patients unaffected brothers. They then reprogrammed these blood cells into stem cells, and these stem cells were converted into neurons in a petri dish. As a result, they obtained neurons that were representative of those from CS patients, and they used these neurons to test treatments.

Morrow who directs the Center for Translational Neuroscience at the Carney Institute for Brain Science and the Brown Institute for Translational Science said the team also used a new gene-editing approach that employs CRISPR-Cas9 technologies to correct patient mutations back to a healthy gene sequence.

CS is caused by a mutation in a gene encoding for NHE6, a protein that helps regulate acid levels within cell structures called endosomes. Past research suggests that the loss of NHE6 causes endosomes to become overly acidic, which disrupts the abilities of developing neurons to branch out and form connections in the growing brain.

Loss of this important protein can arise from a variety of gene mutations in patients. The majority of CS mutations are called nonsense mutations, which prevent NHE6 from being produced at all; four of the five CS patients involved in this study exhibited this class of mutation. However, some CS patients exhibit missense mutations. Individuals with missense mutations still have some NHE6, but it is produced in smaller amounts, and the protein fails to function as it should.

The research team tested two main forms of treatment on the stem-cell-derived neurons: first, gene transfer, which involves adding a healthy NHE6 gene into the cell; and second, administration of trophic factors, which are substances that promote neuron growth and encourage neurons to develop connections with other neurons. The researchers found that the neurons response to treatment depended on the class of mutation present.

The gene transfer studies, which may represent the first steps toward developing gene therapy, were successful in neurons with nonsense mutations. After the researchers inserted a functional NHE6 gene into nonsense-mutation CS neurons, the neurons branched out properly. In neurons with missense mutations, however, gene transfer failed completely. Further tests suggested that the abnormal NHE6 produced as a result of missense mutations may interfere with normal NHE6, thereby rendering gene transfer therapy ineffective in patient cells with these mutations.

In contrast, administration of trophic factors, such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), successfully promoted proper branching in all the CS neurons studied, regardless of mutation type.

While these initial results are encouraging, Morrow hopes that future studies will examine these treatments in animal models.

Our results provide an initial proof-of-concept for these treatment strategies, indicating that they should be studied further, he said. However, we may ultimately need to pay close attention to the class of mutation that a patient has when we choose a specific treatment.

In addition to Morrow, the research team included scientists from Brown University, the University of South Carolina and the Icahn School of Medicine at Mount Sinai. The study was supported by multiple grants from the National Institutes of Health as well as a number of awards from foundations and academic institutions.

This news story was authored by contributing science writerKerry Benson.

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Neurons from patient blood cells enable researchers to test treatments for genetic brain disease - Brown University

Scientists in the Perelman School of Medicine have uncovered the molecular causes of a congenital form of dilated cardiomyopathy (DCM), an often-fatal heart disorder.

This inherited form of DCMwhich affects at least several thousand people in the United States at any one time and often causes sudden death or progressive heart failureis one of multiple congenital disorders known to be caused by inherited mutations in a gene called LMNA. The LMNA gene is active in most cell types, and researchers have not understood why LMNA mutations affect particular organs such as the heart while sparing most other organs and tissues.

In a study published in Cell Stem Cell, the Penn Medicine scientists used stem cell techniques to grow human heart muscle cells containing DCM-causing mutations in LMNA. They found that these mutations severely disrupt the structural organization of DNA in the nucleus of heart muscle cellsbut not two other cell types studiedleading to the abnormal activation of non-heart muscle genes.

Were now beginning to understand why patients with LMNA mutations have tissue-restricted disorders such as DCM even though the gene is expressed in most cell types, says study co-senior author Rajan Jain, an assistant professor of cardiovascular medicine and cell and developmental biology at the Perelman School of Medicine.

This story is by Sophie Kluthe. Read more at Penn Medicine News.

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Stem cell study illuminates the cause of an inherited heart disorder | Penn Today - Penn Today