Sarit Schwartz

Feedback Suppression of PI3Kα Signaling in PTEN-Mutated Tumors Is Relieved by Selective Inhibition of PI3Kβ

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

Interferon α/β Enhances the Cytotoxic Response of MEK Inhibition in Melanoma

Drugs that inhibit the MAPK pathway have therapeutic benefit in melanoma, but responses vary between patients, for reasons that are still largely unknown. Here we aim at explaining this variability using pre- and post-MEK inhibition transcriptional profiles in a panel of melanoma cell lines. We found that most targets are context specific, under the influence of the pathway in only a subset of cell lines. We developed a computational method to identify context-specific targets, and found differences in the activity levels of the interferon pathway, driven by a deletion of the interferon locus. We also discovered that IFNα/β treatment strongly enhances the cytotoxic effect of MEK inhibition, but only in cell lines with low activity of interferon pathway. Taken together, our results suggest that the interferon pathway plays an important role in, and predicts, the response to MAPK inhibition in melanoma. Our analysis demonstrates the value of system-wide perturbation data in predicting drug response.

Targeting wild-type KRAS -amplified gastroesophageal cancer through combined MEK and SHP2 inhibition

The role of KRAS, when activated through canonical mutations, has been well established in cancer1. Here we explore a secondary means of KRAS activation in cancer: focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas2–4. KRAS-amplified gastric cancer models show marked overexpression of the KRAS protein and are insensitive to MAPK blockade owing to their capacity to adaptively respond by rapidly increasing KRAS–GTP levels. Here we demonstrate that inhibition of the guanine-exchange factors SOS1 and SOS2 or the protein tyrosine phosphatase SHP2 can attenuate this adaptive process and that targeting these factors, both genetically and pharmacologically, can enhance the sensitivity of KRAS-amplified models to MEK inhibition in both in vitro and in vivo settings. These data demonstrate the relevance of copy-number amplification as a mechanism of KRAS activation, and uncover the therapeutic potential for targeting of these tumors through combined SHP2 and MEK inhibition.Amplification of wild-type KRAS in a subset of gastroesophageal tumors drives intrinsic resistance to MAPK inhibition that can be overcome by combined targeting of MEK and SHP2.

Abstract 4774: The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling

The PI3K pathway is dysregulated in many cancers via selective activation of class 1 isoforms. In tumors with deficient PTEN function, signaling is driven by PI3K beta. We show here that a selective inhibitor of PI3K beta inhibits the AKT/mTOR pathway in tumors with defective PTEN function, but is ineffective in those where the pathway is driven by receptor tyrosine kinases. However, inhibition of PI3K signaling by PI3K beta inhibitors is limited by relief of AKT/mTOR dependent feedback and reactivation of IGF1R and other receptors. This results in activation of PI3K alpha and a rebound of PI3K-AKT signaling. This rebound is suppressed and tumor cell inhibition is enhanced with combined inhibition of PI3K alpha and beta. Combined administration of isoform selective PI3K inhibitors may more effectively inhibit the pathway than pan-PI3K inhibitors because of the greater selectivity and decreased off-target toxicity of the former. In PTEN deficient models of prostate cancer, triple therapy with PI3K alpha and beta selective inhibitors combined with a potent androgen receptor inhibitor suppresses the reciprocal feedback activation of both pathways and results in marked (complete) eradication of tumors in vivo. Citation Format: Sarit Schwartz, Brett S. Carver, John Wongvipat, Vanessa Rodrik-Outmezguine, Elisa De Stanchina, Cath Trigwell, Simon Barry, Jose Baselga, Sarat Chandarlapaty, Howard I. Scher, Charles L. Sawyers, Neal Rosen. The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4774. doi:10.1158/1538-7445.AM2014-4774

Author Correction: Targeting wild-type KRAS -amplified gastroesophageal cancer through combined MEK and SHP2 inhibition

In the Supplementary Information originally published with this article, a lane was missing in the β-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.

Abstract 735: HER2 protein quantification in multiple cancer indications identifies candidates for HER2 targeted therapies

Background: Anti-HER2 therapy in cancer indications other than breast and gastric cancers is the subject of ongoing clinical trials. We previously used mass spectrometry to identify HER2 protein expression level cutoffs that correlate with standard measures of HER2 positivity (740 and 750 amol/µg in breast and gastric cancers, respectively). These studies also demonstrated that trastuzumab-treated patients whose tumors express high levels of HER2 (≥ 2200 and ≥ 1825 amol/µg in breast and gastric cancers, respectively) survived much longer than patients with lower HER2 levels. We hypothesized that targeted proteomics would reveal high HER2 levels in indications other than breast and gastric cancers, thus identifying patients likely to benefit from anti-HER2 therapy. Methods: We summarized results from samples processed in our CAP/CLIA-certified laboratory. Tumor areas from FFPE tissue blocks (N=3828) representing multiple cancer indications were marked by a pathologist, microdissected, and solubilized to tryptic peptides. In each liquefied tumor sample, HER2 and other protein targets were quantified with mass spectrometry-based proteomic analysis. Results: HER2 superexpression (> 2200 amol/µg) was found in 0.64% (12/1891) of patients with non-breast, non-gastroesophageal cancers. Among indications with > 50 patients tested, the highest rates of HER2 superexpression were in gynecological cancers (3/124; 2.4%) and bladder cancer (1/51; 2.0%). Treatment and outcome data are largely unavailable, but we are aware of 3 anecdotes: In a 74-year-old male with invasive adenocarcinoma of the gallbladder who had disease progression on gemcitabine + cisplatin, proteomic testing found high HER2 protein expression (3105 amol/µg). The patient responded to trastuzumab + FOLFIRI for 5 months. In a uterine cancer patient whose HER2 status was equivocal by genomic analysis, proteomics found high HER2 expression (4995 amol/µg). Proteomics also revealed the absence of a resistance marker for taxane (TUBB3) and high levels of the response marker for antifolate agents (FRalpha = 10500 amol/µg). The patient responded to trastuzumab + taxol for 9 months before developing resistance and responding to trastuzumab + antifolate. Lastly, a cervical cancer patient whose disease had progressed on chemotherapy showed HER2 superexpression (11322 amol/µg). She was treated with anti-HER2 combinations for > 12 months. Conclusions: Patients with high HER2 protein expression as measured by targeted mass spectrometry in multiple cancer types have benefitted from anti-HER2 therapy. Only small numbers of patients with non-breast, non-gastric tumors have HER2 protein levels indicative of survival benefit from anti-HER2 therapy. However, multiplexed targeted proteomics offers simultaneous, precise quantification of other biomarkers (eg, ERCC1, TUBB3, FRalpha) to guide therapy selection for multiple cancer types. Citation Format: Shankar Sellappan, Sarit Schwartz, Ellen Wertheimer, Fabiola Cecchi, Steven W. Mamus, Daniel VT Catenacci, Todd Hembrough. HER2 protein quantification in multiple cancer indications identifies candidates for HER2 targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 735. doi:10.1158/1538-7445.AM2017-735

Abstract P1-17-02: Adaptive and acquired mechanisms of resistance to PI3K inhibitors

Background: Activation of PI3K alpha is among the most frequent oncogenic events in breast transformation, commonly occurring through mutations in the p110 alpha subunit of PI3K or amplification of the HER2 receptor tyrosine kinase. Therapeutic targeting of activated PI3K signaling has been made feasible by the development of highly selective and potent ATP competitive inhibitors of p110 alpha some of which are in late stage clinical development. Unfortunately, clinical response to these compounds has not been seen for the majority of patients and when response occurs it is often short-lived. We have hypothesized that relief of feedback regulation of PI3K signaling constitutes a major mechanism for tumor cell adaptation to inhibitors of oncogenes such as PI3K. Methods: To understand the basis for resistance to PI3K inhibitors we studied the consequences of acute and prolonged PI3K inhibition in patient samples as well as in cell line and murine models of breast cancer. We examined the effects of selective inhibition of PI3K alpha on upstream and parallel signaling pathway components utilizing several phosphoproteomic methods including mass spectrometry, antibody microarrays, and immunoblotting. We further investigated the consequences of PI3K inhibition upon the genome using next generation sequencing. Results: In the context of tumors with high levels of RTK signaling such as HER2+ breast cancers, inhibitors of PI3K alpha led to only transient inhibition of PI3K/AKT signaling with reactivation of signaling closely coinciding in time with loss of feedback suppression of RTKs such as HER3 and IGF1R. This reactivation of AKT signaling could be blocked using inhibitors of the induced RTKs and this was associated with a marked increase in tumor cell death. A second mechanism of reactivation of PI3K signaling was observed in examining the genomes of tumors where inhibition of PI3K alpha was associated with acquired inactivating mutations in PTEN. Loss of PTEN in such tumors was associated with failure of the PI3K alpha inhibitor to block PI3K/AKT signaling but sensitivity to combined PI3K alpha plus PI3K beta inhibition in laboratory models. Finally, in the context of hormone dependent tumor models, inhibition of PI3K alpha was associated with an increase in estrogen receptor activation. This was observed as increases in ER protein expression, ER phosphorylation, and ER binding to established ER target promoters. Combined inhibition of ER and PI3K alpha was demonstrated to be synergistic in these models in vivo. Conclusions: Feedback suppression of upstream and parallel signaling pathways poses a major limitation to the antitumor effects of single agent PI3K alpha inhibition. Combination approaches to potently inhibit PI3K alpha, PI3K beta, and either ER or HER3/IGF1R may prove more effective and durable in the clinic. Citation Format: Sarat Chandarlapaty, Maurizio Scaltriti, Marie Will, Zhiqiang Li, Ana Bosch-Campos, Sarit Schwartz, Vanessa Rodrik-Outmezguine, Michael Berger, Baselga Jose, Neal Rosen. Adaptive and acquired mechanisms of resistance to PI3K inhibitors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-17-02.