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PI3Kβ is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kβ and its coupling to SHIP2 to produce PI(3,4)P(2). We now test whether selectivity for PI3Kβ is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kβ is inhibited, short chain diC8-PIP(3) rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P(2) is SHIP2-independent. Surprisingly, expression of either activated PI3Kβ or PI3Kα mutants rescued the effects of PI3Kβ inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP(3) conversion to PI(3,4)P(2) for invadopodia function, and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer-related deaths, despite accounting for only 10%–15% of total breast cancer cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Targeting UBA1 with the first in-class UBA1 inhibitor TAK-243 induced unresolvable ER-stress and activating transcription factor 4 (ATF4)-mediated upregulation of pro-apoptotic NOXA, leading to cell death. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. In an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. Lastly, c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. We posit UBA1 is an important new target in TNBC expressing high levels of c-MYC.SignificanceGenomic screening of TNBC cell lines revealed broad sensitivity to depletion of the E1 ubiquitin enzyme, UBA1. Disrupting UBA1 with the first in-class inhibitor TAK-243 in TNBC models induces ER-stress through an ATF4-NOXA axis that is dependent on c-MYC, leading to apoptosis, in vitro and in vivo, primary tumor growth inhibition and metastatic inhibition.

Triple negative breast cancer (TNBC) confers the poorest prognosis of the major subtypes of breast cancers, and often inflicts young, African-American and Hispanic women. Steadily improving survival in other subsets of breast cancers are attributed to the implementation of effective targeted therapies, which are non-chemotherapy drugs that target a specific gene in a cancer. For instance, hormone positive BCs are treated effectively with anti-hormone targeted therapy, and HER2 amplified BCs are treated effectively with anti-HER2 targeted therapy. The CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system, which enables specific gene editing to occur throughout the genome, allows for broad and unbiased discovery of new drug targets. We took advantage of this system to ask whether it may identify new druggable targets to treat TNBC and performed whole genome CRISPR/cas9 screening in two TNBC cell lines, BT549 and MDA-MB-468. To account for the heterogeneity found in TNBC, we further expanded the screen into seven other TNBC cell lines, by focusing on a select number of hits from the initial screen. This helped us identify a novel druggable target in TNBC, the ubiquitin activating enzyme 1 (UBA1). We took advantage of a recently reported small molecule inhibitor of UBA1, TAK-243, which is already in clinical trial evaluation in hematological cancers. Characterization of TAK-243 in cell culture models of TNBC and ex-vivo patient-derived xenografts (PDXs) demonstrated nanomolar IC50 toxicity, which was significantly lower than for tissue-derived normal cells. Mechanistically, TAK-243 therapy induced ER stress and the unfolded protein response in TNBC both in vitro and in vivo, leading to upregulation of activation transcription factor 4 (ATF4) and ATF6. Blocking ATF4 led to loss of NOXA and TAK-243 efficacy. TAK-243 induced tumor regressions in two PDX models of TNBC. Overall, these data demonstrate UBA1 is a novel drug target in TNBC. Citation Format: Sheeba Jacob, Tia H. Turner, Ann K. Yu, Colin M. Coon, Mohammad A. Alzubi, Charles T. Jakubik, Ynes Bouck, Mikhail G. Dozmorov, Sosipatros A. Boikos, Jennifer Koblinski, Joshua C. Harrell, Cyril H. Benes, Carlotta Costa, Anthony C. Faber. Genomic screening reveals UBA1 as a potent and druggable target in diverse models of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-28.

The combination of CDK4/6 inhibitors with anti-estrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Since PTEN loss also causes resistance to PI3Kα-inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment sequencing strategies. Citation Format: Carlotta Costa, Ye Wang, Amy Ly, Yasuyuki Hosono, Ellen Murchie, Charlotte S. Walmsley, Tiffany Huynh, Christopher Healy, Rachel Peterson, Shogo Yanase, Charles T. Jakubik, Laura E. Henderson, Leah J. Damon, Daria Timonina, Ioannis Sanidas, Christopher J. Pinto, Mari Mino-Kenudson, James Stone, Nicholas J. Dyson, Leif W. Ellisen, Aditya Bardia, Hiromichi Ebi, Cyril H. Benes, Jeffrey A. Engelman, Dejan Juric. PTEN loss mediates clinical cross-resistance to CDK4/6 and PI3Ká inhibitors in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1903.

Macropinocytosis is an actin-dependent but clathrin-independent endocytic process by which cells nonselectively take up large aliquots of extracellular material. Macropinocytosis is used for immune surveillance by dendritic cells, as a route of infection by viruses and protozoa, and as a nutrient uptake pathway in tumor cells. In this study, we explore the role of class I phosphoinositide 3-kinases (PI3Ks) during ligand-stimulated macropinocytosis. We find that macropinocytosis in response to receptor tyrosine kinase activation is strikingly dependent on a single class I PI3K isoform, namely PI3Kβ (containing the p110β catalytic subunit encoded by PIK3CB). Loss of PI3Kβ expression or activity blocks macropinocytosis at early steps, before the formation of circular dorsal ruffles, but also plays a role in later steps, downstream from Rac1 activation. PI3Kβ is also required for the elevated levels of constitutive macropinocytosis found in tumor cells that are defective for the PTEN tumor suppressor. Our data shed new light on PI3K signaling during macropinocytosis, and suggest new therapeutic uses for pharmacological inhibitors of PI3Kβ.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. Significance: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because PTEN loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting. This article is highlighted in the In This Issue feature, p. 1

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression., Graphical Abstract, Highlights • Amplified MYCN is synthetic lethal with the BCL-2 inhibitor ABT-199 in neuroblastoma • MYCN upregulates the MCL-1 inhibitor, NOXA • MYCN-amplified neuroblastomas are further sensitized to ABT-199 with MLN8237 • ABT-199 with MLN8237 induce tumor regressions in MYCN-amplified neuroblastoma mice, Ham et al. show that MYCN-amplified neuroblastomas are sensitive to treatment with the BCL-2 inhibitor ABT-199 due to MYCN-driven increase of NOXA. Combination treatment with the Aurora Kinase A inhibitor MLN8237 and ABT-199 is synergistic in xenograft models of this tumor type, in part via reducing MCL-1.

Endometrial cancer is the most common gynecologic malignancy in the developed world. These cancers harbor the highest rates of PI3K pathway alterations reported, with PTEN being the most frequently altered gene. Differently from other cancer types, in endometrial cancer a specific hotspot mutation in the lipid phosphatase domain of PTEN is present at residue 130. This hotspot mutation significantly co-occurs with other mutations in the PI3K pathway, such as PIK3CA and PIK3R1. Interestingly, in the TCGA data we also found a positive correlation between this mutation and the expression level of the estrogen receptor alpha. Although it has been reported that the PTEN-R130 mutant loses its lipid phosphatase activity in vitro, little is known regarding the other unconventional functions of PTEN, such as its protein phosphatase activity. We performed unbiased phospho-proteomics experiments in order to explore the impact of this specific mutation in multiple endometrial cancer cell models. Citation Format: Ye Wang, Charles Jakubik, Rachel Peterson, Cyril Benes, Wilhelm Haas, Jeffrey Engelman, Carlotta Costa. Characterization of a hotspot PTEN mutation in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5502.

SummaryBYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110a), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110β isoform. Importantly, the reactivation of PI3K mediated by p110β does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110β inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers.