Extended data related to Figure 3.

Extended data related to Figure 7.

EZH2 and MEK inhibitors do not cause toxicity in vivo.

Extended data related to Figure 5.

Extended data related to Figure 1.

Extended data related to Figure 6.

Abstract Current treatments for KRAS-mutant colorectal cancers are often limited by cellular plasticity and rewiring responses. Here we describe a promising therapeutic strategy that simultaneously targets epigenetic and oncogenic signals. Specifically, we show that inhibitors of histone methyltransferase, EZH2, synergize with various rat sarcoma virus (RAS) pathway inhibitors and promote dramatic tumor regression in vivo. Together these agents cooperatively suppress Wingless and Int-1 (WNT)-driven transcription and drive colorectal cancers into a more differentiated cell state by inducing the Groucho/transducin-like enhancer corepressor, TLE4, along with a network of WNT pathway inhibitors and intestinal differentiation proteins. However, these agents also induce the proapoptotic protein BCL2 modifying factor (BMF), which subsequently kills these more differentiated cells. Accordingly, cell death can be prevented by activating β-catenin, by blocking differentiation, or by ablating BMF expression. Collectively, these studies reveal a new therapeutic approach for treating KRAS-mutant colorectal cancers and illustrate a critical convergence of EZH2 and RAS on oncogenic WNT signals, intestinal differentiation, and apoptosis. Significance: Combined EZH2 and RAS pathway inhibitors kill KRAS-mutant colorectal cancer cells and promote durable tumor regression in vivo. These agents function by cooperatively suppressing the WNT pathway, driving differentiation, and epigenetically reprogramming cells to permit the induction of apoptotic signals, which then kill these more differentiated tumor cells.

Abstract Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We previously showed that the RasGAP DAB2IP is a tumor and metastasis suppressor in breast cancer. Interestingly, we have now generated robust data demonstrating that the loss of DAB2IP also mediates therapeutic resistance in HER2+ breast cancer. First, we genetically ablated DAB2IP in multiple HER2+ breast cancer cell lines and performed manual counting experiments after 6 days of HER2 TKI treatment. In all cell lines, DAB2IP knockdown conferred resistance to HER2 inhibitors. Since HER2 inhibitors are known to induce both apoptosis and senescence, we next investigated how DAB2IP loss specifically affects these phenotypes. Interestingly, DAB2IP knockdown prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Mechanistically, stable knockdown of DAB2IP reduced the suppression of the AKT and ERK pathways and the induction of the pro-apoptotic protein Bim upon HER2i treatment. In addition, cell cycle progression pathways were enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. Interestingly, the levels of these proteins were restored and regrowth in low density assays was inhibited with co-treatment with an NF-kB inhibitor. We next evaluated the relevance of DAB2IP loss in mediating HER2 inhibitor resistance in a SUM190 orthotopic xenograft model. Importantly, while control tumors regressed upon lapatinib treatment, DAB2IP-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. Further understanding the pathways deregulated by DAB2IP loss upon HER2i treatment will be essential for developing effective therapeutic strategies to combat resistance induced by DAB2IP loss. Citation Format: Naiara Perurena, Amy Schade, Natalie Pilla, Patrick Loi, Carrie Rodriguez, Alycia Gardner, Karen Cichowski. Loss of the tumor and metastasis suppressor RasGAP DAB2IP mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B072.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy; however, responses are typically short lived1,2. Thus, there is an urgent need to develop more effective treatments. Components of the PI3K pathway represent plausible therapeutic targets; more than 70% of TNBCs have alterations in PIK3CA, AKT1 or PTEN3-6. However, in contrast to hormone-receptor-positive tumours, it is still unclear whether or how triple-negative disease will respond to PI3K pathway inhibitors7. Here we describe a promising AKT-inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors synergize with agents that suppress the histone methyltransferase EZH2 and promote robust tumour regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. Once TNBCs are differentiated, these agents kill them by hijacking signals that normally drive mammary gland involution. Using a machine learning approach, we developed a classifier that can be used to predict sensitivity. Together, these findings identify a promising therapeutic strategy for this highly aggressive tumour type and illustrate how deregulated epigenetic enzymes can insulate tumours from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.

While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy, however responses are typically short-lived. Thus, there is an urgent need to develop more effective treatments. PI3K pathway components represent plausible therapeutic targets, as approximately 40% of TNBCs have PIK3CA/AKT1/PTEN alterations. However, unlike hormone receptor-positive tumors, it is still unclear if or how PI3K pathway inhibitors will be effective in triple-negative disease. Here we identify a promising AKT inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors potently synergize with agents that suppress the histone methyltransferase, EZH2, and promote robust tumor regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. More importantly, once differentiated, these agents kill TNBCs by hijacking signals that normally drive mammary gland involution. Together these findings identify a promising therapeutic strategy for this highly aggressive tumor type and illustrate how deregulated epigenetic enzymes can insulate tumors from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit. Citation Format: Amy Schade, Naiara Perurena, Marina Watanabe, Carrie L. Rodriguez, Patrick Loi, Natalie Pilla, Rachel A. Davis, Kaia Mattioli, Dongxi Xiang, Jason J. Zoeller, Zhe Li, Ana C. Garrido-Castro, Sara Tolaney, Karen Cichowski. AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-11.

Despite the success of KRAS G12C inhibitors in non–small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to cotarget RAS and mTOR pathways; however, toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identifying the most therapeutically important eukaryotic initiation factor 4F complex–translated (eIF4F-translated) targets. Here, we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.

Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We recently discovered that two emerging tumor suppressor RasGAPs, DAB2IP and RASAL2, cooperatively drive metastatic breast cancer when lost or inactivated. Interestingly, we have now generated robust data demonstrating that the loss of these RasGAPs also induces resistance to HER2 inhibitors in breast cancer. First, we genetically ablated both RASAL2 and DAB2IP in multiple HER2+ breast cancer cell lines (SKBR3, EFM192A, SUM190, BT474) and performed manual counting experiments after 6 days of TKI (lapatinib, tucatinib) treatment. In all cell lines, RASAL2/DAB2IP knockdown conferred resistance to HER2 inhibitors. Moreover, loss of these RasGAPs prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Next, we sought to investigate the individual contribution of each RasGAP to these phenotypes. Surprisingly, we found that RASAL2 and DAB2IP functioned quite differently in this context. Specifically, while RASAL2 loss prevented apoptosis, DAB2IP loss prevented irreversible cell cycle arrest (measured by functional long-term experiments and EdU staining assays). Mechanistically, RASAL2 loss uniquely impaired BIM induction at both mRNA and protein levels, which is required for lapatinib-induced cell death of HER2+ cancer cells. By contrast, cell cycle progression pathways were uniquely enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. These data suggest that RASAL2 and DAB2IP (loss) mediate resistance to HER2 inhibitors by differentially deregulating unique pathways/phenotypes. We next evaluated the relevance of these findings in a SUM190 orthotopic xenograft model. Importantly, while control tumors (expressing both RASAL2 and DAB2IP) regressed upon lapatinib treatment, DAB2IP- and RASAL2-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. These data suggest that the unique phenotypes/pathways induced by both RASAL2 and DAB2IP are important mediators of resistance to HER2 inhibitors. Further understanding the contribution of these pathways to anti-HER2 resistance and determining how RASAL2 and DAB2IP differentially function will be essential for developing effective therapeutic strategies to bypass each type of resistance. Citation Format: Naiara Perurena, Natalie Pilla, Amy Schade, Marina Watanabe, Patrick Loi, Carrie L. Rodriguez, Alycia M. Gardner, Karen Cichowski. Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-06.

Cellular processes are wide and varied: feedback loops, signaling and metabolic pathways, cell cycle progres-sions, to name a few. In this sixth installment of the Special series: Scientific figure development, recent TIBS authors share how they craft these complex processes as informative, yet accessible, figures. In doing so, they consider the questions: what aspects do you consider when generating such a figure? How do you decide how to represent the cellular process being described (i.e., shapes of proteins, cellular content, arrows, etc.)? What program(s) do you prefer for generating such figures and why? Contributing to this article are Szymon W. Kmiecik, first author of 'Molecular mechanisms of heat shock factor 1 regulation' [1] (Figure 5, for example); Qiaoni Shi and Ye-Guang Chen, authors of 'Regulation of Dishevelled protein activity and stability by post -translational modifications and autophagy' [2] (Figure 1); Jannis Moormann and Tatjana M. Hildebrandt, the first and corresponding authors of 'News about amino acid metabolism in plant-microbe interactions' [3] (Figure 1 and others); Amy Schade and Martin Fischer, the first authors and co-corresponding author of 'Coordinating gene expression during the cell cycle' [4] (Figures 3 and 4); and Kyusik Kim, first author of 'Canary in a coal mine: collided ribosomes as sensors of cellular conditions' [5] (Figure 1 and others).

Cell cycle-dependent gene transcription is tightly controlled by the retinoblastoma (RB):E2F and DREAM complexes, which repress all cell cycle genes during quiescence. Cyclin-dependent kinase (CDK) phosphorylation of RB and DREAM allows for the expression of two gene sets. The first set of genes, with peak expression in G1/S, is activated by E2F transcription factors (TFs) and is required for DNA synthesis. The second set, with maximum expression during G2/M, is required for mitosis and is coordinated by the MuvB complex, together with B-MYB and Forkhead box M1 (FOXM1). In this review, we summarize the key findings that established the distinct control mechanisms regulating G1/S and G2/M gene expression in mammals and discuss recent advances in the understanding of the temporal control of these genes.

To identify genes whose loss confers resistance to CHK1 inhibitors, we perform genome-wide CRISPR-Cas9 screens in non-small-cell lung cancer (NSCLC) cell lines treated with the CHK1 inhibitor prexasertib (CHK1i). Five of the top six hits of the screens, MYBL2 (B-MYB), LIN54, FOXM1, cyclin A2 (CCNA2), and CDC25B, are cell-cycle-regulated genes that contribute to entry into mitosis. Knockout of MMB-FOXM1 complex components LIN54 and FOXM1 reduce CHK1i-induced DNA replication stress markers and premature mitosis during Late S phase. Activation of a feedback loop between the MMB-FOXM1 complex and CDK1 is required for CHK1i-induced premature mitosis in Late S phase and subsequent replication catastrophe, indicating that dysregulation of the S to M transition is necessary for CHK1 inhibitor sensitivity. These findings provide mechanistic insights into small molecule inhibitors currently studied in clinical trials and provide rationale for combination therapies.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.