Faculty Bibliography

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

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

Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.

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