BYL719, 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.
•p110α inhibition leads to reactivation of PI3K signaling via p110β•Restoration of PI3K is reliably determined by measuring PIP3 levels, not p-AKT•Dual inhibition of p110α and β blocks rebound of PIP3 and induces tumor regressions
Costa et al. show that PI3Kα inhibition only briefly blocks PI3K signaling even in responsive cancer cells due to PI3Kβ activation. Thus, combined PI3Kα and PI3Kβ inhibition provides greater antitumor efficacy. The authors also find PIP3 to be a better marker of active PI3K signaling than phosphorylated AKT.
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
, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition
and
. Mechanistically,
loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because
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
loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting.
.
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.
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•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.
BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid ...tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death ( BIM ) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.
Significance Small-cell lung cancer (SCLC) is an aggressive carcinoma with few effective treatment options beyond first-line chemotherapy. BH3 mimetics, such as ABT-263, promote apoptosis in SCLC cell lines, but early phase clinical trials demonstrated no significant clinical benefit. Here, we examine the sensitivity of a large panel of cancer cell lines, including SCLC, to ABT-263 and find that high Bcl2-interacting mediator of cell death (BIM) and low myeloid cell leukemia 1 (MCL-1) expression together predict sensitivity. SCLC cells relatively resistant to ABT-263 are sensitized by TORC1/2 inhibition via MCL-1 reduction. Combination of ABT-263 and TORC1/2 inhibition stabilizes or shrinks tumors in xenograft models, in autochthonous SCLC tumors in a genetically engineered mouse model, and in a patient-derived xenograft SCLC model. Collectively, these data support a compelling new therapeutic strategy for treating SCLC.
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
). 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.
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
. 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
rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P
is SHIP2-independent. Surprisingly, the 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
conversion to PI(3,4)P
for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.
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)P2. 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‐PIP3 rescues gelatin degradation in a SHIP2‐dependent manner; rescue by diC8‐PI(3,4)P2 is SHIP2‐independent. Surprisingly, the 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 PIP3 conversion to PI(3,4)P2 for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.
GPCR activation of PI3Kβ drives invadopodia‐mediated matrix degradation by breast cancer cells, via PI3Kβ coupling to SHIP2 to produce PI(3,4)P2. Surprisingly, expression of either activated PI3Kβ or PI3Kα rescues the effects of pertussis toxin or TGX221, which inhibit PI3Kβ. In both cases, matrix degradation is SHIP2‐dependent. Thus, selectivity for PI3K isotypes is obviated by mutational activation of PI3K signaling.
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.
Part 1. Metastasis is the most common cause of death in cancer patients with solid tumors. Metastatic tumor cells invade and metastasize using invadopodia, which are actin-rich structures that permit ...cancer cells to degrade the extracellular matrix. We have previously shown that in triple negative breast cancer cell lines, loss of signaling by PI3Kβ, but not other PI3K isotypes, inhibits integrin signaling, matrix degradation, and experimental metastasis. PI3Kβ produces PI(3,4,5)P3 (PIP3) from PI(4,5)P2, which is converted to PI(3,4)P2 by the SH2 domain containing inositol polyphosphate 5'-phosphatase 2 (SHIP2). Our lab showed that PI3Kβ signals downstream from integrins and is critical for the production of PI(3,4)P2 in invadopodia, which leads to the recruitment of invadopodial proteins such as lamellipodin. We tested whether the selective requirement for PI3Kβ during invadopodia-mediated matrix degradation is preserved under conditions of mutational or biochemical increases in PIP3 production. In MDA-MB-231 triple negative breast cancer (TNBC) cells treated with PI3Kβ inhibitors, short chain diC8-PIP3 rescued gelatin degradation in a SHIP2-dependent manner, whereas rescue of gelatin degradation by diC8-PI(3,4)P2 was SHIP2-independent. Surprisingly, expression of activated mutants of either PI3Kβ or PI3Kα rescued the effects of PI3KB inhibition; gelatin degradation in both cases was SHIP2 dependent. These data confirm the requirement for PIP3 conversion to PI(3,4)P2 for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.Part 2. The ability of tumor cells to invade neighboring tissues, intravasate into blood vessels, and extravasate and colonize at secondary sites, requires interactions with macrophages. Previously, our lab has found that bone marrow-derived macrophages (BMMs) from WT mice, but not KKDD (GBY uncoupled mutant) mice, stimulate tumor cell invasion, IL8 secretion, and transendothelial migration. We now show that macrophages stimulate gelatin degradation by tumor cells, and this stimulation requires PI3Kβ signaling in the macrophages. Recent studies from other labs have suggested that macrophage activation of tumor cell Notch and NF-κβ signaling enhances tumor cell invasiveness. Interestingly, we find that activation of tumor cell NF-κβ is unaffected by mutation of macrophage PI3Kβ, suggesting that defects in this pathway cannot explain the inability of KKDD macrophages to stimulate gelatin degradation. Experiments testing the role of macrophage activation of tumor cell Notch are in progress. We have also performed RNAseq analysis on tumor cell/BMM co-cultures to identify the mechanism(s) by which macrophage PI3Kβ induces tumor cell gelatin degradation by tumor cells. Subsequent studies will validate RNAseq results. We will use these data to design experiments that will rescue macrophage-induced tumor cell gelatin degradation in co-cultures with KKDD BMMs.
Abstract
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.
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