Faculty Bibliography

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.

Introduction: Myeloproliferative neoplasms (MPN) are chronic leukemi-as with dysregulated JAK2 signaling. We hypothesized that dual targeting of ERK1 and ERK2 could enhance control of the MPN clone by preventing MAPK pathway activation.
Method(s): We genetically targeted ERK1 and ERK2 in MPN by combining Jak2V617F with ERK1/ERK2 knockout alleles and hematopoiesis-specific Mx-Cre. To assess engrafment and competitive ftness of the MPN clone, CD45.2 Jak2V617F bone marrow (BM) +/-ERK1/ERK2 double KO (dKO) was competitively transplanted with CD45.1 Jak2 WT BM.
Result(s): Loss of ERK1/ERK2 in primary Jak2V617F mice moderated splenomegaly and excessive erythropoiesis including red cells, reticulocytes and erythroid progenitors. Emergence of leukocytosis was prevented. ERK1/ERK2 loss reduced thrombopoiesis with reduced megakaryocyte progenitors and platelets. Hematopoietic stem/progenitor compartments were reduced and myeloid colony formation diminished in ERK1/ERK2 defcient Jak2V617F mice suggesting a reduced disease-initiating population. In JAK2 mutant vs. WT competitive settings, the Jak2V617F clone was signifcantly reduced by ERK1/ERK2 loss in peripheral blood, BM, myeloid and erythroid progenitors. Myeloid colonies from competitively transplanted mice were mainly Jak2 WT with signifcantly lower contribution of Jak2V617F ERK1/ERK2 dKO cells as compared to settings with intact ERK. Polyglobulia and leukocytosis were normalized and BM fbrosis was prevented in recipients of Jak2V617F ERK1/ERK2 dKO BM. Secondary recipients showed near-complete ablation of the Jak2V617F clone upon ERK1/ERK loss along with normalization of blood counts and spleen size. ERK1/ERK2 deletion combined with Jak2 inhibition with rux-olitinib enhanced therapeutic efficacy with pronounced reductions of the MPN clone and correction of the MPN phenotype.
Conclusion(s): ERK1/ERK2 loss abrogates the competitive ftness of the MPN clone by restricting stem/progenitor compartments and blunting clone expansion 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 enhanced therapeutic strategy in MPN

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

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.

High-grade serous ovarian cancer (HGSOC) is the most common and deadly subtype of ovarian epithelial cancer and is known for its aggressiveness, high recurrence rate, metastasis to other sites, development of resistance to conventional chemotherapy, and general lack of response to immune checkpoint inhibitors. The absence of genomically relevant, immune-competent HGSOC models represents a major barrier to developing new therapies. Taking advantage of a mouse fallopian tube organoid system that we developed, along with lentiviral gene transduction and/or CRISPR/Cas9 technology, we generated multiple new HGSOC models containing combinations of mutations seen in human HGSOC, including homologous recombination (HR)-proficient (Tp53-/-;Ccne1amp;Akt2ampand Tp53-/-;Ccne1amp;Krasamp) and -deficient (Tp53-/-;Brca1-/-;Pten-/-and Tp53-/-;Brca1-/-;Mycamp), and poorly characterized (Tp53-/-;Pten-/-;Nf1-/-) models. These models differ in proliferation, differentiation, and polarity/organoid structure in vitro, as well as tumorigenic capacity and behavior upon orthotopic injection into syngeneic mice. Organoids bearing different mutational spectra show differential sensitivity to conventional HGSOC chemotherapies, signal transduction inhibitors and DDR inhibitors, and evoke distinctly different immune microenvironment in vivo. In particular, the immune microenvironment induced by HRdeficient tumors shows more T-cell infiltration/Treg cells, whereas HR-proficient lines show lower T-cell infiltration but higher levels of myeloid-derived suppressor cells and macrophages. The results of these studies suggest novel, genotype-informed combination therapies for this devastating disease

Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) DNA repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombinationdeficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNASeq (scRNASeq) transcriptomic, and immunofluorescence imaging analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease