Faculty Bibliography

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.

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

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

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.

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.

Members of the PP2A family of serine/threonine phosphatases are important human tumor suppressor genes. Unlike most tumor suppressors, they are rarely mutated/deleted, but rather are impaired by "inhibitor proteins." Two papers in this issue of Cell show how some phenothiazine derivatives reactivate specific PP2A isozymes with potential benefit in cancer and other diseases.

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) 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 recombination-deficient 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 RNA sequencing, and transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and regulatory compartments between HR-proficient and -deficient tumors within the primary and metastatic tumors themselves 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

High-grade serous ovarian cancer (HGSOC) is the most common, deadly subtype of ovarian epithelial cancer. HGSOC typically presents at an advanced stage, with widespread peritoneal metastasis. Surgical debulking and platinum/taxane-based chemotherapy can result in complete responses, but disease almost always recurs, eventually in drug-resistant form. The recent addition of Avastin and PARP inhibitors benefits a subset of patients, yet survival has improved marginally in the past 30 years. Responses to immune checkpoint inhibitors have been disappointing. Genomic studies (e.g., TCGA) show that HGSOC is a complex, diverse disease with widespread copy number abnormalities affecting multiple pathways. Nevertheless, most studies aimed at developing new therapies treat HGSOC as a single entity. The absence of genetically relevant, immune-competent HGSOC models poses a major barrier to developing new therapies. By exploiting 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 the human disease, including homologous recombination (HR)-proficient (Tp53-/-;Ccne1OE;Akt2OE and Tp53-/-;Ccne1OE;KrasOE) -deficient (Tp53-/-;Brca1-/-;Pten-/-and Tp53-/-; Brca1-/-;MycOE and unclassified (Tp53-/-;Pten-/-;Nf1-/-) models. These cells differ in proliferation, differentiation, and polarity/organoid structure in vitro, as well as tumorigenicity and metastatic spread upon orthotopic injection into syngeneic mice. Organoids with different mutations show differential sensitivity to current HGSOC drugs and evoke distinctly different immune microenvironments in vivo. Brca1-/- and CcneOE tumors show significant T-cell infiltration/Treg cells; the latter also have increased myeloid-derived suppressor cells (MDSCs). Pten-/- tumors have lower T-cell infiltration but high levels of MDSCs and macrophages. To test the utility of this platform for new therapeutic development, we focused on CcneOE tumors, because human CCNE-amplified HGSOC responds poorly to current therapy. CcneOE organoids were sensitive to gemcitabine (GEM), probably due to CCNE-evoked replication stress. GEM treatment reduced pro-MDSC cytokines in organoid-conditioned media, and decreased tumor burden in mice. Consistent with these in vitro data, residual tumors had decreased MDSCs (CD11b+Ly6CloLy6Ghi), but persistent Treg (CD24+CD25+FoxP3+) and "exhausted" CD8 T cells (CD8+PD1+). Intriguingly, a combination regimen aimed at attacking tumor cells (gemcitabine) and the residual microenvironment (CTLA4 and PDL1 antibodies) produced complete responses in mice with widespread metastatic disease. Our results argue that genotype-informed combination therapies will be necessary for better outcomes for this devastating disease