Cancer type–specific mutations are common, and their stepwise alteration has been shown for tumors including melanoma and colorectal cancer. Hodis and colleagues started with primary human melanocytes and used precise genome editing to introduce up to five sequential mutations occurring in melanoma. Nine unique melanoma models were generated by introduction of mutations affecting six different pathways: CDKN2A (RB pathway), BRAF (MAPK), TERT (telomerase), PTEN (PI3K/AKT), TP53 (p53), and APC (WNT). In these models, melanocyte genotypes were linked to phenotypes including proliferation, cancer-associated gene expression programs in vitro, as well as rapid tumor growth, tumor pigmentation, and metastasis in vivo. Notably, the authors demonstrated that the melanoma-specific mutations also affected the composition of the tumor microenvironment. Importantly, the melanoma models mimicked gene expression programs found in patient tumors.Expert Commentary: Sequential introduction of cancer type–specific mutations using genome editing may be a powerful approach to study progressive cancer development.Hodis E, Torlai Triglia E, Kwon JYH, Bincalani T, Zakka LR, Parkar S, et al. Stepwise-edited, human melanoma models reveal mutations' effect on tumor and microenvironment. Science 2022;eabi8175. doi: 10.1126/science.abi8175.Oncolytic viruses (OV) are promising biotherapeutics, engineered to selectively propagate and kill only tumor cells. Although OVs are advancing into clinical trials, OV as a monotherapy frequently fails in glioblastoma (GBM). Using a dual-step screening of FDA-approved drugs, Xiao and colleagues found that the CDK4/6 inhibitor could cooperate with OVs. They observed that CDK4/6 inhibition promoted both tumor selective replication of OVs and cytotoxic DNA damage stress in GBM cells, leading to direct tumor cell death. Combination treatment with CDK4/6 inhibitor and VSVΔ51 significantly inhibited GBM growth and prolonged the survival of tumor-bearing mice. CDK4/6 inhibition and VSVΔ51 also synergistically reshaped the tumor microenvironment and enhanced tumor-specific immunity in vivo.Expert Commentary: This study proposes that CDK4/6 inhibition enhances oncolytic therapy by potentiating oncolysis and antitumor immunity in GBM. CDK4/6 inhibitors may provide a therapeutic benefit when combined with oncolytic viruses in the treatment of GBM.Xiao J, Lian J, Fan J, Hou P, Li X, Zhang H, et al. CDK4/6 inhibition enhances oncolytic virus efficacy by potentiating tumor-selective cell killing and T cell activation in refractory glioblastoma. Cancer Research; Published OnlineFirst July 22, 2022; doi: 10.1158/0008-5472.CAN-21-3656.Mutations in the VHL gene underlie the Von Hippel–Lindau (VHL) hereditary cancer predisposition syndrome, with some of these tumors being linked to dysregulated HIF2α while the mechanism driving others is unclear. Li and colleagues performed a comparative proteomic screen using wildtype or VHL mutated tumors, identifying that a majority of proteins downregulated in mutant tumors were mitochondrial proteins. Among the top hits was transcription factor A, mitochondria (TFAM), a master regulator of mitochondrial transcription. Mechanistically, the authors showed that VHL binds TFAM upon hydroxylation by EGLN3, preventing degradation of TFAM by the mitochondrial protease LONP1. The proteosomal inhibitor bortezomib could inhibit LONP1 and restore TFAM and mitochondrial biogenesis. The authors also hypothesized that decreased mitochondrial content in VHL tumors might contribute to therapy resistance. In a therapy-resistant renal carcinoma xenograft, restoration of TFAM and mitochondrial biogenesis sensitized to sorafenib, a kinase inhibitor used in kidney cancers.Expert Commentary: These authors find a novel role of VHL in regulating TFAM and mitochondrial biogenesis and show that pharmacological methods to reactivate mitochondria can sensitize therapy resistant VHL tumors.Li S, Li W, Yuan J, Bullova P, Wu J, Zhang X, et al. Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel–Lindau syndrome. Nat Metab 2022;4:739–58.KRAS is mutated in >90% of pancreatic ductal adenocarcinoma (PDAC) cases, and co-alteration of TP53 indicates worse overall survival for patients. Datta and colleagues showed that co-alteration of both genes was associated with increased infiltration of Ly6G+ granulocytic cells and macrophages, but reduced CD8 T-cell infiltration, suggesting an immune contribution to poor outcome. Single-cell RNA sequencing of KRAS only and KRAS and TP53 co-mutant genetically engineered mouse models of PDAC demonstrated upregulation of CXCL1 in co-mutated tumor cells, driving chemotaxis of Ly6G+ granulocytes. TNF expression from granulocytes recruited to the tumor microenvironment drove a unique immunoregulatory transcriptional program (IRP), an aggressive, stem-like, phenotype in tumor cells. An IRP signature in human PDAC samples was associated with worse prognosis in response to chemotherapy.Expert Commentary: Co-mutation of KRAS and TP53 in PDAC leads to the establishment of an immunoregulatory program in recruited immune cells that drive a stem-like phenotype in neoplastic cells.Datta J, Bianchi A, De Castro Silva I, Deshpande NU, Cao LL, Mehra S, et al. Distinct mechanisms of innate and adaptive immune regulation underlie poor oncologic outcomes associated with KRAS-TP53 co-alteration in pancreatic cancer. Oncogene 2022;41:3640–54.In order to understand how hemopoietic stem cells (HSC) respond to stress signals or infection, Li and colleagues investigated the role of the interferon-signaling mediator STAT1 in HSCs. STAT1 knockout mice harbored increased numbers of immunophenotypic HSCs but fewer functional HSCS. Single-cell transcriptomic analysis of STAT1-KO HSCs showed loss of classical MHCII genes, whose expression was thought to be restricted to professional antigen-presenting cells. Indeed, mouse and human HSCs express MHCII genes. MHCII expression is readily detected on the cell surface of HSCs. MHCIIhi HSC population is lost in STAT1-KO mice, and these MHCIIhi expressing HSCs represent a quiescent subset, displaying reduced self-renewal and repopulation of the myeloid lineage, and specifically reduced megakaryocytic differentiation. MHCIIlo and MHCIIhi HSCS were assessed in a mouse model of a myeloproliferative neoplasm. The MHCIIlo HSCs were responsible for the increased megakarypoiesis and neoplastic expansion.Expert Commentary: STAT1 contributes to HSC heterogeneity, driving an MHCII-expressing HSC population with distinct functionality. STAT1 may be an important gene for the interplay between HSCs and the adaptive immune system.Li J, Williams MJ, Park HJ, Bastos HP, Want X, Prins DP, et al. STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resists myeloablation and neoplastic expansion. Blood; Published online June 29, 2022; doi: 10.1182/blood.2021014009.Neuroblastoma (NB), a pediatric extracranial cancer, is receptive to immunotherapy because it overexpresses the ganglioside GD2 that can be targeted. Mabe and colleagues found that GD2 expression was decreased in vivo and in vitro in mesenchymal NBs and that genetic induction of an adrenergic-to-mesenchymal transition reduced GD2 levels. Mechanistically, reduced GD2 levels were found due to decreased expression of ST8SIA1, a key enzyme involved in GD2 synthesis. Overexpression of ST8SIA1 restored GD2 expression in mesenchymal NB. Reduced expression of ST8SIA1 in mesenchymal NB was associated with increased H3K27me3 modifications at the locus. EZH2 inhibition with tazemetostat restored GD2 expression and increased sensitivity to anti-GD2 therapies in vivo. Importantly, EZH2 inhibition, while increasing GD2 on NB cells, minimally increased GD2 levels on normal tissues, reducing the risk of toxicities associated with anti-GD2-based therapies.Expert Commentary: Mesenchymal states of NB are resistant to anti-GD2 immunotherapy because of reduced GD2 synthesis, and sensitivity can be restored by EZH2 inhibition.Mabe NW, Huang M, Dalton GN, Alexe G, Schaefer DA, Geraghty AC, et al. Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nature Cancer; Published online July 11, 2022; doi: 10.1038/s43018-022-00405-x.Note: Breaking Insights are written by Cancer Research editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.