Amy Young

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL–selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2High/BCL-XLLow. In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132–44. ©2016 AACR.

KRAS Allelic Imbalance Enhances Fitness and Modulates MAP Kinase Dependence in Cancer

Investigating therapeutic outliers that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D knockin mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.

The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer.

Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer.

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity. Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging. Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes. Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561–72. ©2017 AACR.

Abstract S6-04: The PI3K inhibitor, taselisib, has enhanced potency in PIK3CA mutant models through a unique mechanism of action

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies. Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented. Citation Format: Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action. [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 370.

Abstract P4-06-05: Treatment ofESR1mutant andPIK3CAmutant patient-derived breast cancer xenograft models reveals differential anti-tumor responses to estrogen receptor degraders and PI3K inhibitorsin vivo

The phosphoinositide 3-kinase (PI3K) pathway is a key driver of hormone receptor (HR)–positive breast cancer growth and survival. It is estimated that 40-45% of HR+ breast cancers harbor oncogenic mutations in the PIK3CA gene, which encodes the p110α isoform of PI3K. Taselisib (GDC-0032) is a mutant-selective PI3K inhibitor that demonstrates enhanced potency in PIK3CA mutant breast cancer cells and is being developed as a treatment for metastatic breast cancer that targets PIK3CA-mutant, HR-positive, HER2-negative patients. Activating mutations in the ESR1 gene were recently described in metastatic breast cancer. These mutations confer hormone independent growth and may be associated with resistance to aromatase inhibitors. Drugs that selectively bind and antagonize the Estrogen Receptor alpha (ERα) protein and target it for degradation, such as fulvestrant, are referred to as selective estrogen receptor degraders (SERDs). Preclinical activity of the orally bioavailable SERD, GDC-0810, has not been well characterized in ESR1 mutant PDX models. Therefore, our aim was to evaluate the efficacy and pharmacodynamic responses to agents that target ERα and PI3K as monotherapies and in combination, in ESR1 and PIK3CA mutant HR+ breast cancer patient-derived xenograft (PDX) models. We hypothesized that mutational status of ESR1 and PIK3CA may predict the responsiveness of HR+ PDX models to SERDs and PI3K inhibitors in vivo. Characterization of seven PDX models included authentication of hormone receptor status by immunohistochemistry (IHC) and determination of ESR1 and PIK3CA genotype and allele frequency by exome sequencing. For a subset of models that utilize estrogen for growth, mice were supplemented with 17β-estradiol, and cells or tumor fragments were implanted into the fat pad of intact female NOD-SCID or NOD-SCID-IL2Rgamma null mice and treated with fulvestrant, GDC-0810, or taselisib. Both fulvestrant and GDC-0810 were efficacious in ESR1 wild type (WT) and mutant PDX models but to variable degrees ranging from tumor stasis to growth delay, with GDC-0810 resulting in superior single agent activity at relevant clinical exposure in the WHIM20 and WHIM43 ESR1 mutant models. PIK3CA mutations (E542K, E545K, M1004V, and H1047R) were confirmed in six PDX models and PI3K pathway activation verified by strong pS6RP IHC staining. Taselisib induced tumor growth inhibition and tumor regressions in models harboring PIK3CA mutations, and models with no detectable expression of WT p110α were the most sensitive. In the WHIM43 (ESR1 D538G, PIK3CA M1004V), HCI-011 (ESR1 WT, PIK3CA E545K) and HCI-013 (ESR1 Y537S, PIK3CA H1047R) PDX models, combining fulvestrant and taselisib treatment further enhanced tumor growth inhibition with respect to either treatment alone. Our studies demonstrate the diverse anti-tumor responses of HR+ PDX models to SERDs and the PI3K inhibitor taselisib in the context of clinically relevant ESR1 and PIK3CA mutations. Pharmacological and genomic characterization of additional PDX models may aid in strengthening associations between genotype, drug sensitivity and predictive biomarkers of response. Citation Format: Young A, Crocker L, Cheng E, Lacap J, Hamilton P, Oeh J, Ingalla E, Arrazate A, Hager J, Nannini M, Friedman L, Daemen A, Giltnane J, Sampath D. Treatment of ESR1 mutant and PIK3CA mutant patient-derived breast cancer xenograft models reveals differential anti-tumor responses to estrogen receptor degraders and PI3K inhibitors in vivo [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-05.

Abstract 156: Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming “hotspot” mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations. Citation Format: Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development [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 156. doi:10.1158/1538-7445.AM2017-156

Abstract 146: The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models

Activating mutations in PIK3CA are commonly found in a wide variety of human cancers, and the dysregulation of the phosphoinositide-3 kinase (PI3K) signaling pathway has been implicated in tumor cell growth and survival. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent in cancer cells bearing PIK3CA mutants than those with wildtype PIK3CA. Preclinical studies demonstrate that taselisib induces more apoptotic cell death in PIK3CA mutant cancer cells than other PI3K inhibitors. We have discovered that taselisib has a dual mechanism of action, both blocking PI3K signaling, and inducing a decrease in p110a protein levels. Mass spec analysis reveals that taselisib treatment leads to the specific depletion of mutant p110alpha without significant change in wildtype p110alpha protein levels. This drug-induced p110a protein depletion is rescued by E1 inhibitors and by proteasome inhibitors. Other clinical PI3K inhibitors, including p110alpha selective and pan-PI3K inhibitors, are unable to induce the depletion of mutant p110 alpha protein. Furthermore, we have discovered that taselisib more effectively maintains pathway suppression in PIK3CA mutant cells at 24 hrs in response to feedback. In comparison to other clinical-stage PI3K inhibitors administered at a maximum tolerated dose, taselisib has superior efficacy with increased tumor regressions in PIK3CA mutant xenograft models. In summary, these preclinical studies indicate that PI3K inhibitors, which have the ability to trigger degradation of mutant p110a protein, can more effectively suppress the signaling pathway, which may result in greater anti-tumor activity and improved therapeutic index in PIK3CA mutant tumors. Citation Format: Kyung W. Song, Kyle A. Edgar, Donald S. Kirkpatrick, Lilian Phu, Stephen Schmidt, Michelle Nannini, Rebecca Hong, Eric Cheng, Lisa Crocker, Amy Young, Deepak Sampath, Lori Friedman. The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models [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 146. doi:10.1158/1538-7445.AM2017-146

Abstract 2672: Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis; thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The current study investigates preclinical mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of PI3K alpha sparing inhibition of PI3K beta. In preclinical studies, taselisib demonstrates greater potency in cancer cell lines harboring activating PI3K alpha mutations vs wild-type lines. Taselisib induces tumor growth arrest and regressions at tolerated doses in xenograft and patient-derived xenograft (PDX) tumor models bearing PI3K alpha mutations. Notably, taselisib is distinguished from other PI3K inhibitors by enhanced potency in PI3K alpha mutant isogenic cells compared to parental cells. Pathway inhibition and apoptosis are associated with the enhanced activity of taselisib in PI3K alpha mutant cells. Other PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not achieve the same level of activity in PI3K alpha mutant cell lines. Taselisib is more effective at maintaining suppression of the signaling pathway upon activation of Receptor Tyrosine Kinases (RTKs), such as after alleviation of negative feedback within the pathway. Additionally, a screen to evaluate secreted factors contributing to pan-PI3K inhibitor resistance was carried out in SW48 parental and PI3Kα H1047R isogenic cells. Growth factor ligands for the EGFR family were found to reduce the activity of PI3K inhibitors. Taken together, taselisib offers greater preclinical activity in PI3K alpha mutant cells when compared to other PI3K inhibitors, which may indicate a wider therapeutic index. Citation Format: Kyle A. Edgar, Michelle Nannini, Rebecca Hong, Charlie Eigenbrot, Stephen Schmidt, Amy Young, Deepak Sampath, Jeffrey J. Wallin, Lori S. Friedman. Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models. [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 2672. doi:10.1158/1538-7445.AM2015-2672