HHi reverses the CA-MSC driven tumor immune cell exclusion restoring the response to ICI.

Rationale : Aldehyde dehydrogenase (ALDH) enzymes are often upregulated in cancer cells and associated with therapeutic resistance. ALDH enzymes protect cells by metabolizing toxic aldehydes which can induce DNA double stand breaks (DSB). We recently identified a novel ALDH1A family inhibitor (ALDHi), 673A. We hypothesized that 673A, via inhibition of ALDH1A family members, could induce intracellular accumulation of genotoxic aldehydes to cause DSB and that ALDHi could synergize with inhibitors of the ATM and ATR, proteins which direct DSB repair. Methods : We used immunofluorescence to directly assess levels of the aldehyde 4-hydroxynonenal and comet assays to evaluate DSB. Western blot was used to evaluate activation of the DNA damage response pathways. Cell counts were performed in the presence of 673A and additional aldehydes or aldehyde scavengers. ALDH inhibition results were confirmed using ALDH1A3 CRISPR knockout. Synergy between 673A and ATM or ATR inhibitors was evaluated using the Chou-Talalay method and confirmed in vivo using cell line xenograft tumor studies. Results : The ALDHi 673A cellular accumulation of toxic aldehydes which induce DNA double strand breaks. This is exacerbated by addition of exogenous aldehydes such as vitamin-A (retinaldehyde) and ameliorated by aldehyde scavengers such as metformin and hydralazine. Importantly, ALDH1A3 knockout cells demonstrated increased sensitivity to ATM/ATR inhibitors. And, ALDHi synergized with inhibitors of ATM and ATR, master regulators of the DSB DNA damage response, both in vitro and in vivo. This synergy was evident in homologous recombination (HR) proficient cell lines. Conclusions : ALDHi can be used to induce DNA DSB in cancer cells and synergize with inhibitors the ATM/ATR pathway. Our data suggest a novel therapeutic approach to target HR proficient ovarian cancer cells.

Cancer associated MSCs (CA-MSCs) are multipotent stromal cells that generate tumor desmoplasia and promote tumor growth. Here, we investigated the impact of CA-MSCs on the ovarian tumor immune microenvironment. In vitro and in vivo analyses confirmed that CA-MSCs secrete numerous cytokines and chemokines, including Ccl2, Cx3cl1 and Tgfb1, that are known to induce the recruitment of monocytes into the tumor and promote macrophage immunosuppressive phenotype. Using a syngeneic and immune responsive murine ovarian tumor model, we found that CA-MSCs restrict the intra-tumoral trafficking of CD8+ T cells and inhibit the response to immune checkpoint inhibitor therapy. Similarly, in patient samples the presence of CA-MSC is inversely correlated with CD8+ T cell infiltration into tumor islets. Single cell RNAseq analyses revealed that tumoral monocytes and macrophages of a-PD-L1 treated-CA-MSCs enriched tumors express high levels of the protein transforming growth factor beta induced (Tgfbi). In ovarian cancer patient samples TGFBI is primarily expressed in the desmoplastic stroma and correlated with poor prognosis. Importantly, we found that hedgehog signaling inhibitor (HHi) therapy reversed CA-MSC driven tumor desmoplasia, reduced the number of tumor-associated myeloid cells and decreased their expression of Tgfbi. These events restored the infiltration and activation of CD8+ T cells into the tumor islets and response to a-PDL1 therapy. We are currently evaluating the role of Tgfbi in the regulation of innate and adaptive immune response. Our findings demonstrated CA-MSCs critically regulate anti-tumor immunity and support HHi as an adjunct to anti-PDL1 therapy to increase repose to immune therapy in ovarian cancer.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Supported by Ovarian Cancer Research Alliance