Faculty Bibliography

High-grade serous tubo-ovarian carcinoma (HGSC) is a major cause of cancer-related death. Treatment is not uniform, with some patients undergoing primary debulking surgery followed by chemotherapy (PDS) and others being treated directly with chemotherapy and only having surgery after three to four cycles (NACT). Which strategy is optimal remains controversial. We developed a mathematical framework that simulates hierarchical or stochastic models of tumor initiation and reproduces the clinical course of HGSC. After estimating parameter values, we infer that most patients harbor chemoresistant HGSC cells at diagnosis and that, if the tumor burden is not too large and complete debulking can be achieved, PDS is superior to NACT due to better depletion of resistant cells. We further predict that earlier diagnosis of primary HGSC, followed by complete debulking, could improve survival, but its benefit in relapsed patients is likely to be limited. These predictions are supported by primary clinical data from multiple cohorts. Our results have clear implications for these key issues in HGSC management.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess-triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow-derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of p53, Brca1, Pten, Nf1, and overexpression of Myc and p53R172H, which was contrasted to an identical model carrying wild-type Brca1. For homologous recombination-proficient tumors, we constructed genotypes combining loss of p53, and overexpression of Ccne1, Akt2, p53R172H, and driven by KRASG12V or Brd4 or Smarca4 overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC.

The paucity of genetically informed, immune-competent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube epithelial organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high grade serous tubo-ovarian carcinoma (HGSC) models exhibiting mutational combinations seen in HGSC patients. Detailed analysis of homologous recombination (HR)-proficient (Tp53-/-;Ccne1OE;Akt2OE ;KrasOE), HR-deficient (Tp53-/-;Brca1-/-;MycOE), and unclassified (Tp53-/-;Pten-/-;Nf1-/-) organoids revealed differences in in vitro properties (proliferation, differentiation, "secretome"), copy number aberrations, and tumorigenicity. Tumorigenic organoids had variable sensitivity to HGSC chemotherapeutics, evoked distinct immune microenvironments that could be modulated by neutralizing organoid-produced chemokines/cytokines. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T-cell dependent responses in Tp53-/-;Ccne1OE;Akt2OE;Kras HGSC; by contrast, Tp53-/-;Pten-/-;Nf1-/- tumors failed to respond. Mouse and human HGSC models showed genotype-dependent similarities in chemosensitivity, secretome, and immune microenvironment. Genotype-informed, syngeneic organoid models could provide a platform for the rapid evaluation of tumor biology and therapeutics.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell mediated anti-tumor immunity but many patients do not respond and a significant proportion develops inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1 triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.

Introduction: Myeloproliferative neoplasms (MPN) are chronic leuke-mias with dysregulated Jak2 signaling. We hypothesized that genetic tar-geting of ERK1/2 could enhance control of the MPN clone by preventing MAPK pathway activation.
Method(s): We genetically targeted ERK1/2 in MPN by introducing ERK1/2 knockout alleles and hematopoiesis-specific Mx-Cre in Jak2V617F mice. To assess engraftment and competitive fitness of the MPN clone, CD45.2 Jak2V617F bone marrow (BM) +/- ERK1^-/- ERK2^fl/fl was competitively transplanted with CD45.1 Jak2 WT BM.
Result(s): Loss of ERK1/2 in Jak2V617F Ba/F3 cells reduced cells growth by 60% and potentiated Jak2 inhibition by ruxolitinib. In MPN mice, it moderated splenomegaly and excessive erythropoiesis including red cells, reticulocytes and erythroid progenitors. Hematopoietic stem/pro-genitor compartments were reduced and myeloid colony formation di-minished in Jak2V617F ERK1^-/- ERK2^fl/fl mice, suggesting reduced dis-ease-initiating cells. In competitive transplants, ERK1/2 loss significantly reduced the Jak2V617F MPN clone in peripheral blood, BM, myeloid and erythroid progenitors. Myeloid colonies emerging from Jak2V617F ERK1^-/- ERK2^fl/fl:WT competitively transplanted mice were predominantly Jak2 WT as compared to settings with intact ERK. Polyglobulia was nor-malized and BM fibrosis prevented in recipients of Jak2V617F ERK1^-/- ERK2^fl/fl BM. ERK1/2 deletion combined with Jak2 inhibition with rux-olitinib enhanced therapeutic efficacy with extensive reduction of the MPN clone and correction of the MPN phenotype.
Conclusion(s): ERK1/2 loss abrogates the competitive fitness of the MPN clone by restricting stem/progenitor compartments and cooperates with JAK2 inhibition resulting in correction of MPN features. Our data suggest targeting of ERK1/ERK2 in combination with JAK2 inhibition as an en-hanced therapeutic strategy in MPN