Ashish Juvekar

Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer

Cancer cells often select for mutations that enhance signalling through pathways that promote anabolic metabolism. Although the PI(3)K/Akt signalling pathway, which is frequently dysregulated in breast cancer, is a well-established regulator of central glucose metabolism and aerobic glycolysis, its regulation of other metabolic processes required for tumour growth is not well defined. Here we report that in mammary epithelial cells, oncogenic PI(3)K/Akt stimulates glutathione (GSH) biosynthesis by stabilizing and activating NRF2 to upregulate the GSH biosynthetic genes. Increased NRF2 stability is dependent on the Akt-mediated accumulation of p21Cip1/WAF1 and GSK-3β inhibition. Consistently, in human breast tumours, upregulation of NRF2 targets is associated with PI(3)K pathway mutation status and oncogenic Akt activation. Elevated GSH biosynthesis is required for PI(3)K/Akt-driven resistance to oxidative stress, initiation of tumour spheroids, and anchorage-independent growth. Furthermore, inhibition of GSH biosynthesis with buthionine sulfoximine synergizes with cisplatin to selectively induce tumour regression in PI(3)K pathway mutant breast cancer cells, both in vitro and in vivo. Our findings provide insight into GSH biosynthesis as a metabolic vulnerability associated with PI(3)K pathway mutant breast cancers.

Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

Significance Mutations in the PI3K pathway are highly prevalent in cancers, and isoform-specific and pan-PI3K inhibitors have entered clinical trials in both solid and hematologic malignancies. The PI3K δ-specific inhibitor idelalisib (in combination with rituximab) was recently approved for the treatment of chronic lymphocytic leukemia. However, identifying tumor types and biological mechanisms that predict for response to PI3K inhibitors as single agents or in combination has been a challenge. Our data indicate that PI3K inhibitors induce DNA damage in tumors that have defects in DNA damage-repair pathways and that they do so by impairing the production of Rib phosphate and amino acids needed for deoxynucleotide synthesis. We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors.

Closing escape routes: inhibition of IL-8 signaling enhances the anti-tumor efficacy of PI3K inhibitors

The phosphoinositide 3-kinase (PI3K) pathway serves as a relay where signals that emanate from the cell membrane are received and converted into intracellular signals that promote proliferation and survival. Inhibitors of PI3K hold promise for the treatment of breast cancer because activation of this pathway is highly prevalent. However, as is increasingly observed with inhibitors of cell signaling, there appear to be mechanisms of primary and secondary resistance. Britschgi and colleagues report that compensatory activation of the IL-8 signaling axis is a mechanism of primary resistance to PI3K inhibitors in some triple-negative breast cancers. In a set of experiments that carefully emulate the clinical scenario in a mouse model, they show that simultaneous inhibition of Janus kinase 2 enhances the efficacy of PI3K/mammalian target of rapamycin inhibition. Their paper lends further support to the concept that successful design of treatments with signal transduction inhibitors must anticipate potential escape routes - and include agents to simultaneously block them.

Abstract CT338: Combination of a PI3K- and a PARP-inhibitor to treat high-grade serous ovarian or triple-negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Clinical trials have shown promising responses of BRCA-linked breast and ovarian cancers to PARP inhibitor therapy, but remissions are often short-lived and incomplete. The PI3K pathway is frequently activated in these malignancies. Recently, we reported in vivo synergy of a PI3K- and a PARP-inhibitor in a mouse model of BRCA1-related breast cancer. While the PARP-inhibitor olaparib alone attenuated tumor growth modestly, a dramatic reduction in tumor growth was observed when olaparib was combined with the PI3K-inhibitor BKM120. In BRCA1-mutant HCC1937 BC cells, PI3K- (but not akt-) inhibition increased indicators of DNA damage, such as poly-ADP-ribosylation and γH2AX, suggesting a critical role of PI3K activity for the maintenance of genomic stability. Here, we report on the molecular mechanism underlying this synergy, on treatment outcomes in an improved mouse model system and the development of an early-phase clinical study. Pre-clinical modeling was done in BRCA1-mutant HCC1937 cells and in a mouse model based on the syngenic transplantation of tumors derived on the K14-Cre BRCA1f/fp53f/f background. Metabolic profiling in vitro and in vivo showed that PI3K-inhibition decreased flux through glycolysis and specifically through the non-oxidative pentose-phosphate pathway, the main source of ribose-5-phosphate required for the de novo synthesis of nucleotides in HCC1937 cells. Within 3 hours and preceding cell cycle changes, BKM120 caused a decrease in nucleotide pools that was further exacerbated by the addition of olaparib. Nucleotide shortage led to to replication stress with the appearance of γH2AX and increased poly-ADP-ribosylation. Tumors with complete loss of BRCA1 and p53 proved particularly vulnerable to this treatment strategy. We were able to induce complete and durable remissions of murine tumors arising on the K14-Cre BRCA1f/fp53f/f background with a 28-day course of BKM120 and Olaparib. These preclinical data have served as the rationale for a phase I, multi-center study ([NCT01623349][1]) combining the oral PARP inhibitor olaparib with the oral PI3-kinase inhibitor BKM120 in patients with recurrent HGSC or recurrent TNBC. The study is being conducted through the Stand Up to Cancer (SU2C)s Targeting PI3-kinase in Womens Cancers Dream Team. It has a 3 + 3 design, escalating if 0/3 or 1/6 participants have a DLT during the first cycle of therapy. The study objectives are to determine the recommended phase II dose (RP2D) of daily continuous oral olaparib (using the tablet formulation) and BKM120, assess toxicities, safety, and preliminary activity of this combination, and determine pharmacokinetic profiles of both agents as well as translational endpoints. The study serves as an example for the development of a clinical trials concept for TNBC and HGSC based on a close collaboration of basic and clinical scientists through the SU2C mechanism. Citation Format: Gerburg M. Wulf, Ashish Juvekar, Costas A. Lyssiotis, Hai Hu, Sina Yadegarynia, Hui Liu, Baek Kim, Eric Winter, Ralph Scully, John Asara, Lewis C. Cantley, Ursula Matulonis. Combination of a PI3K- and a PARP-inhibitor to treat high-grade serous ovarian or triple-negative breast cancer. [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 CT338. doi:10.1158/1538-7445.AM2014-CT338 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01623349&atom=%2Fcanres%2F74%2F19_Supplement%2FCT338.atom

Abstract IA08: PI3K- and PARP-inhibitors for the treatment of women cancers.

Amplification of PIK3CA is highly prevalent in ovarian cancer and isoform-specific and pan-PI3K inhibitors have entered clinical trials. However, identifying tumor subtypes and biological mechanisms that predict for response to PI3K inhibitors as single agents or in combination has been a challenge. We previously reported that combining a phophoinositide 3-kinase (PI3K) inhibitor with a poly ADP ribose polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. We now show that the enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of deoxynucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four deoxynucleotide triphosphates (dATP, dTTP, dGTP and dCTP), while inhibition of AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K-inhibition disproportionately affects the non-oxidative pentose phosphate pathway (non-ox PPP) that delivers ribose-5-posphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer combined PI3K- and PARP-inhibition was superior to either agent alone to induce durable remissions of established tumors. Our data indicate that PI3K inhibitors induce DNA damage in tumors that have defects in DNA damage repair pathways and that they do so by impairing the production of Ribose-5-phosphate and amino acids needed for deoxynucleotide synthesis. An ongoing phase I/II study (NCT01623349, PI: Matulonis) examines the safety and toxicity of a PI3K/Parp-inhibitor combination in ovarian and breast cancer. Citation Format: Gerburg M. Wulf, Ashish Juvekar, Lewis C. Cantley, Ursula A. Matulonis. PI3K- and PARP-inhibitors for the treatment of women cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA08.

Abstract 159: Analysis of resistance to the combination of a PI3K- and Parp-inhibitor using a genomic sequencing approach

The objective of this study is to investigate the genomic and transcriptonal changes that are associated with resistance to treatment with the combination of a PI3K-inhibitor (NVP-BKM120) and a PARP-inhibitor (Olaparib). Previous studies have shown that the combination of a PI3K-inhibitor and a PARP-inhibitor synergistically reduced the growth of BRCA-related xenograft tumors derived from patients with TNBC. However, in clinical practice, primary and secondary resistance to this combination is observed. We hypothesize that acquired resistance is the result of a genomic evolution resulting from the selection pressure exerted by the drug treatment. We used a genetically engineered mouse model, K14-Cre BRCA1f/fp53f/f, where primary tumors had been propagated in Cre-negative littermates and treated to point of resistance with NVP-BKM120 or NVP-BKM120 plus Olaparib. For each of the three tumors, we analyzed exome sequencing and RNA-seq using Illumina technology at the stage of sensitivity (C), resistance to PI3K-inhibition (B) and resistance to the combination (BO); i.e. for each individual tumor a parental clone and its drug-resistant subclones, obtained in vivo, were analyzed. We used the Mutect software tool to call somatic mutations, VarScan2 to call somatic copy number variations, Cuffdiff for differential expression analysis, and TophatFusion. Data were integrated to identify genes for functional analyses using MetaCore and MSigDB software tools. We found that tumors resistant to the combination drug treatment in general had fewer gross genomic alterations (CNVs) than parental tumors, indicative of evolution of a less variable subclone. However, these tumors also displayed a higher number of non-synonymous mutations than their parental clone. Pathway analysis showed that somatically mutated genes in PI3K-inhibitor resistant tumors were highly enriched for antigen processing and presentation, while those of tumors resistant to the combination treatment were enriched for histone modification pathways. Notably, the tumors resistant to PI3K-inhibitor alone were enriched for Insulin-processing pathways. Genomic losses in PI3K-inhibitor treated tumors enriched for the estrogen receptor pathway (ESR) in breast cancer and DNA damage pathways. In conclusion, our experimental design of analyzing isogenic tumors resistant to PI3K- or combined PI3K- and Parp-inhibitors enabled us to identify genomic alterations and pathways that explain the evolution to drug resistance. Citation Format: Sheida Nabavi, Ashish Juvekar, Nicholas Wang, Lewis C. Cantley, Gerburg M. Wulf. Analysis of resistance to the combination of a PI3K- and Parp-inhibitor using a genomic sequencing approach. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 159.

Abstract IA21: Combination treatments that include PI3K-inhibitors for the treatment of triple-negative breast cancer

Alterations in the PI3K pathway are highly prevalent in triple-negative breast and high grade ovarian cancer, including BRCA1- and BRCA2-related disease. On the cellular level, loss of BRCA1 increases genomic instability and reliance on single strand break repair, hence creating an opportunity to treat BRCA-related TNBC or OC with PARP-inhibitors. In addition, loss of BRCA1 and/or p53 lead to relaxation of negative feed-back loops in mitogenic signaling, resulting in highly proliferative malignancies. For these reasons PARP-inhibitor Olaparib and PI3K-inhibitor NVP-BKM120 were tested in combination and found to be synergistic in mouse models of BRCA1-related BC. Surprisingly, PI3K-inhibition enhanced a DNA damage phenotype, caused by a profound decrease in DNA synthesis that could be observed after treatment with NVP-BKM120 in vivo and in vitro. The decrease in DNA synthesis was due to reduced Nucleoside synthesis resulting from a block in glycolysis that led to a drop in flux through the non-oxidative pentose phosphate pathway. In a preclinical mouse model (K14-Cre BRCA1f/fp53f/f), the combination of NVP-BKM120 and Olaparib could induce complete remissions, while PI3K-inhibitor alone marginally slowed disease progression and PARP-inhibitor alone stabilized the disease. This concept was translated into a clinical trial: BKM120/Olaparib for Triple Negative Breast Cancer or High Grade Serous Ovarian Cancer (NCT01623349; PI: Ursula Matulonis). Dose escalation for this study and accrual to an extension cohort at the MTD (NVP-BKM120 50 mg once a day in combination with Olaparib 300 mg twice a day) have been completed and a second dose escalation arm with NVP-BYL719 and Olaparib is ongoing. Citation Format: Gerburg M. Wulf, Ashish Juvekar, Costas M. Lyssiotis, Hai Hu, Kim Baek, Sina Yadegarynia, Ralph Scully, Eric Winer, John Asara, Lewis C. Cantley, Ursula Matulonis. Combination treatments that include PI3K-inhibitors for the treatment of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA21.

Abstract 2650: PI 3-Kinase inhibitors enhance the synthetic lethality of Parp inhibitors

Introduction: Clinical trials have shown promising responses of BRCA-linked breast and ovarian cancers to PARP inhibitor therapy, but remissions are often short-lived and incomplete. Frequent loss of INPP4B and PTEN leads to activation in the PI3K pathway in TNBC, making the PI3K pathway an attractive treatment target. Here we present mechanistic data that explain how PI3K- and PARP-inhibition enhance each other. Methods: We examined the effects of PI3K-inhibitors BKM120 or BYL719 and PARP-inhibitor Olaparib on the metabolism of HCC1937 cells (BRCA1 5382C mutation and homozygous deletion of PTEN and p53) measuring lactic acid production, via seahorse analysis, carbon metabolism using targeted Mass Spectrometry (LC-MS/MS) and DNA synthesis. PI3K- but not Akt- or SGK-inhibition induced a DNA damage response in this BRCA1 mutant cell line as well as in a mouse model of BRCA1-related breast cancer (K14-Cre BRCA1f/f p53f/f) mouse model. Summary: Metabolic profiling showed that PI3K-inhibition decreased flux through glycolysis and specifically through the non-oxidative pentose-phosphate pathway, the main source of ribose-5-phosphate required for the de novo synthesis of nucleotides in BRCA1-mutant breast cancer cells. Nucleotide shortage led to replication stress with the appearance of γH2AX, increased poly-ADP-ribosylation and an acute drop in DNA synthesis in cell cultures as well as in vivo. PI3K inhibition led to a reduced and error-prone S-phase due to a decrease in nucleotide biosynthesis which could be rescued upon exogenous supply of nucleosides. In a mouse model (K14-Cre BRCA1f/fp53f/f induced breast cancers) we could confirm that both BKM120 and BYL719 enhanced the efficacy of Parp-inhibitor Olaparib (median PFS for Olaparib 50 days, for BKM120 4 days, for BYL719 6 days and for both combinations, BKM120+Olaparib and BYL719+Olaparib > 120 days). Conclusion: Preceding cell cycle arrest, PI3K-inhibition leads to a decrease in DNA synthesis due to decreased Ribose-5-phosphate production required for nucleotide synthesis. PI3K-inhibitors lower nucleotide pools, lead to impaired DNA damage repair and S-phase progression and further augment the synthetic lethality of Parp-inhibitors in BRCA1-related breast cancer. Citation Format: Ashish P. Juvekar, Sina Yadegarynia, Hai Hu, Costas A. Lyssiotis, Hui Liu, John M. Asara, Ralph Scully, Lewis C. Cantley, Gerburg M. Wulf. PI 3-Kinase inhibitors enhance the synthetic lethality of Parp inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2015-2650

Abstract 1081: Non-random genomic alterations in BRCA1-related breast cancer

BRCA1-related breast cancer is characterized by a high degree of genomic instability. However, to which extent this instability is a random result of the homologous recombination defect or the selection of the tumor phenotype, is unknown. The objective of this study is to investigate the genomic aberrations (copy number variation and somatic mutations) of BRCA1-related breast cancer, using a mouse model, to identify likely nonrandom aberrations and their biological functions. We examined genomic aberrations of three independent BRCA1-related tumors from a genetically engineered mouse model, K14-Cre BRCA1f/fp53f/f. Tumors had been propagated in Cre-negative littermates and liver tissue of the recipient mice from the controls were used as a germline control. Fresh frozen tumors and normal samples were used for exome sequencing and RNA-seq using Illumina technology. A commonly used workflow, GATK, was used for the sequence alignment, preprocessing and calling somatic mutations. VarScan2 was used for calling somatic copy number variations. We investigate the prevalence of common aberrations among these mouse in human BRCA1-related breast and ovarian cancer from The Cancer Genome Atlas (TCGA). We used the BioMart software tool to map mouse genes to human genes for the cross species analysis. After identifying common aberrant regions and genes across human and mouse we applied enrichment analysis using GOSeq, MetaCore and MSigDB software tools to identify the biological functions of the genetic alterations observed in BRCA1-related breast cancer across species. The analysis of exome sequencing data from BRCA1-related mouse tumors revealed surprisingly similar patterns of CNVs and rates of non-synonymous mutations. Correspondingly, distinct pattern were identified with regard to CNVs in human BRCA1-related breast and ovarian cancer. The cross species analysis of BRCA1-related TCGA ovarian and breast cancer tumors and BRCA1-related mouse tumors identified commonly aberrant regions that mapped to the long arms of human chromosomes 1, 8, 17 and 20 in both breast and ovarian cancer. Functionally, the blocks of genes that were commonly amplified in BRCA1-related breast or ovarian cancer included genes that transduce mitogenic signaling and ligand independent activation of estrogen receptors ESR1 and ESR2 as well as genes of the renin-angiotensin system. To conclude, copy number aberrations in BRCA1-related breast or ovarian cancers are not random and enrich for genes that enforce mitogenic signaling. The data support the notion that in addition to loss of cell cycle checkpoint control, tumor development requires activation of a mitogenic signaling program that could potentially be targeted for treatment. Citation Format: Sheida Nabavi, Kristina M. Holton, Ashish Juvekar, Nicholas Wang, Olivier Elemento, Lewis C. Cantley, Gerburg M. Wulf. Non-random genomic alterations in BRCA1-related breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1081. doi:10.1158/1538-7445.AM2015-1081

Abstract 4781: EBV infection of mammary epithelial cells promotes malignant transformation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Whether the human tumor virus, Epstein-Barr virus (EBV) promotes breast cancers remains controversial and a potential mechanism has remained elusive. Herein we show EBV binds mammary epithelial cells (MECs) via CD21 and upon establishing a persistent infection triggers expansion of MEC progenitor cells (CD24low/CD44high), increases mammosphere formation, blocks differentiation and in conjunction with activated Ras accelerates formation of breast cancers in NOD/SCID mice. Similar to nasopharyngeal carcinoma (NPC), breast cancers derived from EBV-infected and Ras-transduced MECs exhibited a latency II pattern infection (LMP1 high, LMP2A positive) with a significant number of lytic transcripts. A human gene expression signature for “EBVness” was derived from these tumors and application to publicly available data sets of breast cancer patients showed “EBVness” was - associated with high grade (40 vs 13.5%) estrogen-receptor (ER)-negative status (31.8 vs. 10.5%), p53 mutation (37.5 vs 14.5%) and poor survival. Citation Format: Hai Hu, Gerburg Wulf, Ashish Juvekar, Dirk P Dittmer, Joyce Fingeroth. EBV infection of mammary epithelial cells promotes malignant transformation. [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 4781. doi:10.1158/1538-7445.AM2013-4781 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.