Cooperation Between Distinct Cancer Driver Genes Underlies Intertumor Heterogeneity in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: The pattern of genetic alterations in cancer driver genes in patients with hepatocellular carcinoma (HCC) is highly diverse, which partially explains the low efficacy of available therapies. In spite of this, the existing mouse models only recapitulate a small portion of HCC inter-tumor heterogeneity, limiting the understanding of the disease and the nomination of personalized therapies. Here, we aimed at establishing a novel collection of HCC mouse models that captured human HCC diversity. METHODS: By performing hydrodynamic tail-vein injections, we tested the impact of altering a well-established HCC oncogene (either MYC or ß-catenin) in combination with an additional alteration in one of eleven other genes frequently mutated in HCC. Of the 23 unique pairs of genetic alterations that we interrogated, 9 were able to induce HCC. The established HCC mouse models were characterized at histopathological, immune, and transcriptomic level to identify the unique features of each model. Murine HCC cell lines were generated from each tumor model, characterized transcriptionally, and used to identify specific therapies that were validated in vivo. RESULTS: Cooperation between pairs of driver genes produced HCCs with diverse histopathology, immune microenvironments, transcriptomes, and drug responses. Interestingly, MYC expression levels strongly influenced ß-catenin activity, indicating that inter-tumor heterogeneity emerges not only from specific combinations of genetic alterations but also from the acquisition of expression-dependent phenotypes. CONCLUSIONS: This novel collection of murine HCC models and corresponding cell lines establishes the role of driver genes in diverse contexts and enables mechanistic and translational studies.

Palbociclib (PD-0332991), a selective CDK4/6 inhibitor, restricts tumour growth in preclinical models of hepatocellular carcinoma.

OBJECTIVE: Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Palbociclib, a well-tolerated and selective CDK4/6 inhibitor, has shown promising results in the treatment of retinoblastoma (RB1)-positive breast cancer. RB1 is rarely mutated in HCC, suggesting that palbociclib could potentially be used for HCC therapy. Here, we provide a comprehensive characterisation of the efficacy of palbociclib in multiple preclinical models of HCC. DESIGN: The effects of palbociclib on cell proliferation, cellular senescence and cell death were investigated in a panel of human liver cancer cell lines, in ex vivo human HCC samples, in a genetically engineered mouse model of liver cancer, and in human HCC xenografts in vivo. The mechanisms of intrinsic and acquired resistance to palbociclib were assessed in human liver cancer cell lines and human HCC samples by protein and gene expression analyses. RESULTS: Palbociclib suppressed cell proliferation in human liver cancer cell lines by promoting a reversible cell cycle arrest. Intrinsic and acquired resistance to palbociclib was determined by loss of RB1. A signature of 'RB1 loss of function' was found in <30% of HCC samples. Palbociclib, alone or combined with sorafenib, the standard of care for HCC, impaired tumour growth in vivo and significantly increased survival. CONCLUSIONS: Palbociclib shows encouraging results in preclinical models of HCC and represents a novel therapeutic strategy for HCC treatment, alone or particularly in combination with sorafenib. Palbociclib could potentially benefit patients with RB1-proficient tumours, which account for 70% of all patients with HCC.

Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies.

Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase's impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use.

Phenotype-Based Screens with Conformation-Specific Inhibitors Reveal p38 Gamma and Delta as Targets for HCC Polypharmacology.

The approved kinase inhibitors for hepatocellular carcinoma (HCC) are not matched to specific mutations within tumors. This has presented a daunting challenge; without a clear target or mechanism, no straightforward path has existed to guide the development of improved therapies for HCC. Here, we combine phenotypic screens with a class of conformation-specific kinase inhibitors termed type II to identify a multikinase inhibitor, AD80, with antitumoral activity across a variety of HCC preclinical models, including mouse xenografts. Mass spectrometry profiling found a number of kinases as putative targets for AD80, including several receptor and cytoplasmic protein kinases. Among these, we found p38 gamma and delta as direct targets of AD80. Notably, a closely related analog of AD80 lacking p38δ/γ activity, but retaining several other off-target kinases, lost significant activity in several HCC models. Moreover, forced and sustained MKK6 → p38→ATF2 signaling led to a significant reduction of AD80 activity within HCC cell lines. Together with HCC survival data in The Cancer Genome Atlas and RNA-seq analysis, we suggest p38 delta and gamma as therapeutic targets in HCC and an "AD80 inhibition signature" as identifying those patients with best clinical outcomes.

ß-Catenin Activation Promotes Immune Escape and Resistance to Anti-PD-1 Therapy in Hepatocellular Carcinoma.

PD-1 immune checkpoint inhibitors have produced encouraging results in patients with hepatocellular carcinoma (HCC). However, what determines resistance to anti-PD-1 therapies is unclear. We created a novel genetically engineered mouse model of HCC that enables interrogation of how different genetic alterations affect immune surveillance and response to immunotherapies. Expression of exogenous antigens in MYC;Trp53 -/- HCCs led to T cell-mediated immune surveillance, which was accompanied by decreased tumor formation and increased survival. Some antigen-expressing MYC;Trp53 -/- HCCs escaped the immune system by upregulating the ß-catenin (CTNNB1) pathway. Accordingly, expression of exogenous antigens in MYC;CTNNB1 HCCs had no effect, demonstrating that ß-catenin promoted immune escape, which involved defective recruitment of dendritic cells and consequently impaired T-cell activity. Expression of chemokine CCL5 in antigen-expressing MYC;CTNNB1 HCCs restored immune surveillance. Finally, ß-catenin-driven tumors were resistant to anti-PD-1. In summary, ß-catenin activation promotes immune escape and resistance to anti-PD-1 and could represent a novel biomarker for HCC patient exclusion. SIGNIFICANCE: Determinants of response to anti-PD-1 immunotherapies in HCC are poorly understood. Using a novel mouse model of HCC, we show that ß-catenin activation promotes immune evasion and resistance to anti-PD-1 therapy and could potentially represent a novel biomarker for HCC patient exclusion.See related commentary by Berraondo et al., p. 1003.This article is highlighted in the In This Issue feature, p. 983.

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-ß Pathway.

Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or ß3) is regulated by the Polycomb protein CBX7. ß3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor ß (TGF-ß) pathway in a cell-autonomous and non-cell-autonomous manner. ß3 levels are dynamically increased during oncogene-induced senescence (OIS) through CBX7 Polycomb regulation, and downregulation of ß3 levels overrides OIS and therapy-induced senescence (TIS), independently of its ligand-binding activity. Moreover, cilengitide, an αvß3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP) without affecting proliferation. Finally, we show an increase in ß3 levels in a subset of tissues during aging. Altogether, our data show that integrin ß3 subunit is a marker and regulator of senescence.

Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation.

Developing animals frequently adjust their growth programs and/or their maturation or metamorphosis to compensate for growth disturbances (such as injury or tumor) and ensure normal adult size. Such plasticity entails tissue and organ communication to preserve their proportions and symmetry. Here, we show that imaginal discs autonomously activate DILP8, a Drosophila insulin-like peptide, to communicate abnormal growth and postpone maturation. DILP8 delays metamorphosis by inhibiting ecdysone biosynthesis, slowing growth in the imaginal discs, and generating normal-sized animals. Loss of dilp8 yields asymmetric individuals with an unusually large variation in size and a more varied time of maturation. Thus, DILP8 is a fundamental element of the hitherto ill-defined machinery governing the plasticity that ensures developmental stability and robustness.

Intron retention in the Drosophila melanogaster Rieske Iron Sulphur Protein gene generated a new protein.

Genomes can encode a variety of proteins with unrelated architectures and activities. It is known that protein-coding genes of de novo origin have significantly contributed to this diversity. However, the molecular mechanisms and evolutionary processes behind these originations are still poorly understood. Here we show that the last 102 codons of a novel gene, Noble, assembled directly from non-coding DNA following an intronic deletion that induced alternative intron retention at the Drosophila melanogaster Rieske Iron Sulphur Protein (RFeSP) locus. A systematic analysis of the evolutionary processes behind the origin of Noble showed that its emergence was strongly biased by natural selection on and around the RFeSP locus. Noble mRNA is shown to encode a bona fide protein that lacks an iron sulphur domain and localizes to mitochondria. Together, these results demonstrate the generation of a novel protein at a naturally selected site.