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Triple-negative breast cancer (TNBC) is a highly heterogeneous disease with multiple, distinct molecular subtypes that exhibit unique transcriptional programs and clinical progression trajectories. Despite knowledge of the molecular heterogeneity of the disease, most patients are limited to generic, indiscriminate treatment options: cytotoxic chemotherapy, surgery, and radiation. To identify new intervention targets in TNBC, we used large-scale, loss-of-function screening to identify molecular vulnerabilities among different oncogenomic backgrounds. This strategy returned salt inducible kinase 2 (SIK2) as essential for TNBC survival. Genetic or pharmacological inhibition of SIK2 leads to increased autophagic flux in both normal-immortalized and tumor-derived cell lines. However, this activity causes cell death selectively in breast cancer cells and is biased toward the claudin-low subtype. Depletion of ATG5, which is essential for autophagic vesicle formation, rescued the loss of viability following SIK2 inhibition. Importantly, we find that SIK2 is essential for TNBC tumor growth in vivo . Taken together, these findings indicate that claudin-low tumor cells rely on SIK2 to restrain maladaptive autophagic activation. Inhibition of SIK2 therefore presents itself as an intervention opportunity to reactivate this tumor suppressor mechanism.

Tumours frequently activate genes whose expression is otherwise biased to the testis, collectively known as cancer–testis antigens (CTAs). The extent to which CTA expression represents epiphenomena or confers tumorigenic traits is unknown. In this study, to address this, we implemented a multidimensional functional genomics approach that incorporates 7 different phenotypic assays in 11 distinct disease settings. We identify 26 CTAs that are essential for tumor cell viability and/or are pathological drivers of HIF, WNT or TGFβ signalling. In particular, we discover that Foetal and Adult Testis Expressed 1 (FATE1) is a key survival factor in multiple oncogenic backgrounds. FATE1 prevents the accumulation of the stress-sensing BH3-only protein, BCL-2-Interacting Killer (BIK), thereby permitting viability in the presence of toxic stimuli. Furthermore, ZNF165 promotes TGFβ signalling by directly suppressing the expression of negative feedback regulatory pathways. This action is essential for the survival of triple negative breast cancer cells in vitro and in vivo. Thus, CTAs make significant direct contributions to tumour biology., Proteins usually expressed solely in the testes are often found over-expressed in cancer and are termed cancer testis antigens. Here, the authors use a comprehensive screening strategy to identify 26 cancer-testis antigens that promote tumorigenic behaviour.

Our group employed a pangenomic loss of function screening approach to systematically dissect the molecular architecture that functionally supports Triple Negative Breast Cancer (TNBC). Given that treatment options for this disease are typically limited to cytotoxic therapies, we applied a paclitaxel-based synthetic lethal approach to identify those molecular components that modulate chemoresponsiveness. To account for heterogeneity within the patient population, we screened 4 triple negative tumor-derived breast cancer cell lines that represent the spectrum of TNBC oncogenic aberrations and chemoresponse profiles. We also accounted for the two molecular subtypes, Claudin-Low and Basal-Like, that comprise the majority of TNBC cases. These screens revealed a number of core modulators of tumor cell viability and paclitaxel-induced cellular stress that have not been previously appreciated for supporting TNBC biology at the cell autonomous level. For example, this strategy implicated AMPK family member, SIK2, as a key nutrient sensor that may inhibit excessive autophagy. Inhibition of SIK2 leads to enhanced autophagic flux and a loss of cell viability in a variety of TNBC genetic backgrounds. In addition, we discovered the signaling supported by the cytokine receptor, CXCR3, and its ligand, CXCL9, to be repurposed to promote mitotic fidelity and thus, tumor cell fitness specifically in the Basal-Like TNBC molecular subtype. Finally, we uncovered a requirement for the transcription factor, ZNF165, in specifying the gene expression profiles activated by TGFβ signaling. In particular, the TGFβ target protein, SERPINE1, is required to promote prosurvival signaling in the Claudin-Low TNBC molecular subtype. Given that the expression of ZNF165 is otherwise restricted to the male testes, ZNF165 may represent a mechanism by which tumors engage anomaly-expressed proteins to promote survival. Taken together, we have uncovered novel tumor cell vulnerabilities in TNBC that could be therapeutically leveraged to enhance current therapeutic approaches. Citation Format: Kimberly Maxfield, Aleix Prat, Kathleen Corcoran, Angelique Whitehurst. Dissecting the functional landscape of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A27.

Tumors frequently activate genes whose expression is otherwise restricted to testes, and which can invoke a humoral immune response in cancer patients. Given their germline-biased expression pattern, these Cancer-Testes antigens (CT-antigens, CTAs) may afford the potential for an extraordinary therapeutic window, if they are required for tumorigenic behaviors. While the function of CTAs in normal and neoplastic settings remains obscure, mounting evidence suggests that specific CTAs can contribute to tumor cell autonomous behaviors. To generate a more comprehensive understanding of the functional roles of CTAs, we have employed an unbiased, multi-dimensional, loss of function discovery platform to systematically assess the contribution of CT-antigens to a range of tumorigenic signaling pathways and phenotypes. By employing a diverse panel of 11 tumor-derived cell lines to screen 120 CTAs, we find that these tumor-activated genes are required to support a number of the hallmarks of cancer. For example, we reveal multiple MAGE family members that mitigate response to mitotic stress and hypoxia. Additionally, we find that the SPANX family of CTAs contributes to β-catenin signaling. Finally, we reveal a role for GAGE proteins in supporting the viability of a range of tumor types. The diversity of neoplastic settings analyzed in our primary screen allowed for the identification of CTAs that support tumor cell fitness in multiple disease backgrounds. For example, we have uncovered CTAs that contribute to mitochondrial integrity in lung, breast, melanoma and sarcoma. Alternatively, we find that CTAs can display lineage selective dependencies, such as supporting TGFβ signaling in claudin-low breast tumors. Taken together, these observations support the hypothesis that CTAs are frequently integrated into the tumor cell regulatory environment to support cell division, survival and modulate stress responses by regulating key signaling pathways. Thus, CT-antigens present previously unrecognized tumor-specific vulnerabilities that may be exploited for therapeutic intervention. Citation Format: Kathleen Corcoran, Patrick Taus, Rebecca Sinnott, Kimberly Maxfield, Joshua Wooten, Angelique Whitehurst. Framing therapeutic opportunities in tumor-activated gametogenic programs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4379. doi:10.1158/1538-7445.AM2013-4379

Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor patient prognosis. Patients who respond poorly to the current standard of care cytotoxic chemotherapeutic regimens only have about a 55% five-year survival rate due to the lack of other effective TNBC therapeutics. To uncover new therapeutic entry points for TNBC, we employed a pangenomic loss of function siRNA screening approach. To account for heterogeneity within the patient population, we screened 4 triple negative tumor-derived breast cancer cell lines that represent the spectrum of oncogenic aberrations and molecular subtypes in TNBC. By intersecting the datasets from these 4 cell lines, we revealed core modulators of viability and chemotherapy-induced cellular stress that have not been previously implicated in TNBC biology. For example, our screen uncovered a tractable energy-sensing AMPK family member that supports tumor cell viability in a subset of TNBC tumors. We also discovered tumor cell vulnerabilities unique to the Basal-Like and Claudin-Low TNBC molecular subtypes. In particular, Basal-Like tumor cell division is sensitive to perturbations in specific cytokine signaling. With respect to Claudin-Lows, we found an increased sensitivity to depletion of transcriptional modulators and downstream targets of TGFβ signaling, which suggests that TGFβ signaling may not only be required for a mesenchymal phenotype but also for Claudin-Low tumor cell survival. Our findings suggest that a set of tumor cell vulnerabilities can partition TNBC into functional subgroups and these vulnerabilities could lead to new therapeutics improving overall TNBC patient prognosis. Citation Format: Kimberly Maxfield, Aleix Prat, Kathleen Corcoran, Angelique Whitehurst. Dissecting the functional landscape of triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-207. doi:10.1158/1538-7445.AM2013-LB-207