PI3Kα Inhibitor Research Articles

Abstract OT-36-01: Phase III study of GDC-0077 or placebo (pbo) with palbociclib (P) + fulvestrant (F) in patients with PIK3CA-mutant/hormone receptor-positive/HER2-negative locally advanced or metastatic breast cancer (HR+/HER2- LA/MBC)

Abstract Background HR+/HER2- BC is the most common BC subtype, and adjuvant endocrine therapies (ET) are an integral part of its management; however, ~30% of patients still relapse. Development of ET resistance remains a challenge in HR+/HER2- BC; aberrant phosphoinositide 3-kinase (PI3K) signaling contributes to ET resistance and PIK3CA mutations occur in ~40% of HR+/HER2- BCs. Current research has led to the development of new targeted therapies, including CDK4/6 inhibitors and PI3K inhibitors (alpelisib). Preclinical models have demonstrated synergy between CDK4/6 inhibitors and PI3K inhibitors, with PI3K inhibitors blocking CDK4/6 inhibitor resistance development. GDC-0077 is a potent, selective PI3Kα inhibitor and a mutant p110α-degrader with anti-tumor activity alone and in combination with ET + P in PIK3CA-mutant preclinical models. An ongoing phase I trial showed that GDC-0077 + P + F could be combined at maximum doses. INAVO120 is a phase III, randomized, double-blind, pbo-controlled study that will assess efficacy and safety of GDC-0077/pbo + P + F in patients with PIK3CA-mutant/HR+/HER2- LA/MBC (NCT04191499). Trial design Patients are randomized 1:1 to GDC-0077 (9 mg orally; once daily [QD] continuously on a 28-day cycle) + P (125 mg orally; QD, days 1-21 of each 28-day cycle) + F (500 mg intramuscularly every 28 days with a loading dose in Cycle 1) or pbo + P + F. Circulating tumor DNA or tumor tissue must be positive for a PIK3CA mutation. Eligibility Patients whose disease progressed during/within 12 months of adjuvant ET completion, and who have not received prior systemic therapy for LA/MBC. Aims The primary endpoint is investigator-assessed progression-free survival (PFS) using Response Evaluation Criteria in Solid Tumors v1.1. Secondary endpoints include objective response rate (ORR); best overall response (BoR); duration of response (DoR); clinical benefit rate (CBR); overall survival (OS); safety; patient-reported outcomes; and pharmacokinetics. Statistical methods Patients are stratified by visceral disease, primary vs secondary resistance, and geographic region. For the primary PFS analysis, the treatment arms will be compared using a two-sided stratified log-rank test. The treatment effect will be quantified via a hazard ratio, computed from a stratified Cox proportional-hazards regression, including a 95% confidence interval. Median PFS will be estimated using Kaplan-Meier methodology. Accrual Target enrollment is 400 patients at ~210 sites globally. The study is open for enrollment and, as of 06/04/2020, eight patients have been enrolled. Contact information For more information or to refer a patient, email global.rochegenentechtrials@roche.com or call 1-888-662-6728 (USA only). Originally submitted to the ESMO 2020 Virtual Meeting. Citation Format: Nicolas Turner, Komal Jhaveri, Kevin Kalinsky, Sibylle Loibl, Sherene Loi, Seock-Ah Im, Cristina Saura, Peter Schmid, Jennifer L Schutzman, Thomas J Stout, Guiyuan Lei, Katherine E Hutchinson, Eirini Thanopoulou, Dejan Juric. Phase III study of GDC-0077 or placebo (pbo) with palbociclib (P) + fulvestrant (F) in patients with PIK3CA-mutant/hormone receptor-positive/HER2-negative locally advanced or metastatic breast cancer (HR+/HER2- LA/MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-36-01.

Abstract PS5-12: Preliminary correlative analysis of clinical outcomes with PIK3CA mutation (mut) status from a phase I/Ib study of GDC-0077 in patients (pts) with hormone receptor-positive/HER2-negative metastatic breast cancer (HR+/HER2- mBC)

Abstract Background Mutations in p110α, encoded by PIK3CA, are present in ~40% of HR+/HER2- BCs. GDC-0077, a PI3Kα-selective inhibitor and mutant PI3Kα degrader, elicits antitumor activity in PIK3CAmut preclinical models as a single agent and when combined with endocrine therapy (ET). New evidence suggests BCs harboring multiple PIK3CAmut exhibit increased signaling through the PI3K/AKT pathway and are more sensitive to PI3Kα inhibitors compared with BCs with a single PIK3CAmut. We report a preliminary analysis of PIK3CAmut status with clinical outcomes from an ongoing study of GDC-0077 alone or with ET (letrozole/fulvestrant) ± palbociclib (palbo) in pts with PIK3CAmut HR+/HER2- mBC (NCT03006172). Methods Detectable PIK3CAmut from local tumor tissue/blood-based assay or tumor tissue by cobas PIK3CA assay were required to enroll. Plasma-derived circulating tumor (ct) DNA was collected at baseline (BL), cycle 1 day 15 (C1D15), and C2D1 (in the cohort where GDC-0077 starts at C1D15) to detect PIK3CAmut. Paired tumor samples were analyzed for Ki67 and pAKT/pS6 expression by immunohistochemistry. Single vs multiple PIK3CAmut was correlated with the percentage of pharmacodynamic (PD) inhibition of Ki67/pAKT/pS6 expression; with the PIK3CAmut allele frequency ratio between BL and C1D15 or C2D1 (MAFr15); with best overall response (BOR, RECIST v1.1); and with time on treatment (TOT) in days. Statistical analyses: Kruskal-Wallis and Mann-Whitney-Wilcoxon for group and pairwise comparisons, respectively, and two-sample proportion testing for categorical comparisons. Results Data cutoff was 03/20/2020. PIK3CAmut were detected in 87/103 (84.5%) pts with BL ctDNA available for sequencing. Multiple PIK3CAmut were detected in 21/87 (24.1%) BL ctDNA samples: 9 from pts treated with single-agent GDC-0077; 8 from pts treated with GDC-0077 + letrozole/fulvestrant; and 4 from pts treated with GDC-0077 + letrozole/fulvestrant + palbo. The median number of lines of prior therapy for metastatic disease was not different between pts with multiple (3.0 lines) vs single (2.5 lines) PIK3CAmut detected at BL (p = 0.205). Median percentage inhibition of Ki67/pAKT/pS6 expression was greater in pts with multiple (-65.8, -70.3, -66.8%, respectively) vs single (-42.1, -34.1, -29.5%) PIK3CAmut detected at BL (p = 0.095, 0.002, 0.056). Median MAFr15 was lower in pts with multiple (MAFr15 0.01) vs single PIK3CAmut (MAFr15 0.15) detected at BL (p = 0.004). Of 73 pts with both BL ctDNA-detected PIK3CAmut and BOR data, 16/16 (100%) with multiple PIK3CAmut experienced BOR of partial response (PR) or stable disease (SD) while 42/57 (73.7%) with single PIK3CAmut experienced BOR of PR or SD (p = 0.051). No pts with multiple PIK3CAmut detected experienced a BOR of progressive disease. Median TOT was greater in pts with multiple PIK3CAmut (196 days) vs single PIK3CAmut (140.5 days) detected at BL, but this was not significant (p = 0.1804). Conclusions The fraction of pts in which multiple PIK3CAmut were identified from BL ctDNA in this HR+/HER2- mBC dataset (24.1%) was slightly higher than reported elsewhere. This may be due to the method of detection (blood vs tissue) and/or the definition of multiple PIK3CAmut used. Pts in which multiple PIK3CAmut were detected by ctDNA exhibited greater depth of PD biomarker inhibition in tumors and experienced PR/SD more often compared with pts in which only one PIK3CAmut was detected. However, no significant associations were observed with the number of prior lines of therapy for metastatic disease or TOT. The dataset is currently too small to assess the impact of different treatment regimens in this study but will be re-evaluated as the data mature. Citation Format: Komal Jhaveri, Dejan Juric, Andrea Varga, Nicolas Turner, Peter Schmid, Cristina Saura, Mafalda Oliveira, Ian E Krop, Kevin Kalinsky, Antoine Italiano, Erika Hamilton, Valentina Gambardella, Andrés Cervantes, Philippe L Bedard, Bonnie P Liu, Jessica W Chen, Junko Aimi, Stephanie Royer-Joo, Jennifer L Schutzman, Katherine E Hutchinson. Preliminary correlative analysis of clinical outcomes with PIK3CA mutation (mut) status from a phase I/Ib study of GDC-0077 in patients (pts) with hormone receptor-positive/HER2-negative metastatic breast cancer (HR+/HER2- mBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-12.

Combining Neratinib with CDK4/6, mTOR, and MEK Inhibitors in Models of HER2-positive Cancer.

Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor that is FDA approved for HER2-overexpressing/amplified (HER2+) breast cancer. In this preclinical study, we explored the efficacy of neratinib in combination with inhibitors of downstream signaling in HER2+ cancers in vitro and in vivo. Cell viability, colony formation assays, and Western blotting were used to determine the effect of neratinib in vitro. In vivo efficacy was assessed with patient-derived xenografts (PDX): two breast, two colorectal, and one esophageal cancer (with HER2 mutations). Four PDXs were derived from patients who received previous HER2-targeted therapy. Proteomics were assessed through reverse phase protein arrays and network-level adaptive responses were assessed through Target Score algorithm. In HER2+ breast cancer cells, neratinib was synergistic with multiple agents, including mTOR inhibitors everolimus and sapanisertib, MEK inhibitor trametinib, CDK4/6 inhibitor palbociclib, and PI3Kα inhibitor alpelisib. We tested efficacy of neratinib with everolimus, trametinib, or palbociclib in five HER2+ PDXs. Neratinib combined with everolimus or trametinib led to a 100% increase in median event-free survival (EFS; tumor doubling time) in 25% (1/4) and 60% (3/5) of models, respectively, while neratinib with palbociclib increased EFS in all five models. Network analysis of adaptive responses demonstrated upregulation of EGFR and HER2 signaling in response to CDK4/6, mTOR, and MEK inhibition, possibly providing an explanation for the observed synergies with neratinib. Taken together, our results provide strong preclinical evidence for combining neratinib with CDK4/6, mTOR, and MEK inhibitors for the treatment of HER2+ cancer.

Discovery of 2-(5-(quinolin-6-yl)-1,3,4-oxadiazol-2-yl)acetamide derivatives as novel PI3Kα inhibitors via docking-based virtual screening

PI3Kα is an attractive target for PIK3CA mutated malignant tumor and searching for lead compounds with novel scaffold is important for the development of PI3Kα inhibitors. Therefore, the strategy of docking-based virtual screening was performed to discovery potent inhibitors. The 4L2Y_A PI3Kα crystal structure was used as the model protein receptor due to its high docking reliability. After the multistep virtual screening protocol and biological evaluation, three hits were picked up and further similarity searching led to more potent 2-(5-(quinolin-6-yl)-1,3,4-oxadiazol-2-yl)acetamide derivatives ES-25 and ES-27. In addition, the primary SAR of these novel derivatives was discussed, which provide a basis for the further structural modification.

Preliminary Results from a Phase II Study of Response-Adapted Therapy with Copanlisib and Rituximab for Untreated Follicular Lymphoma

Introduction Follicular lymphoma (FL) has a highly variable clinical course. Chemoimmunotherapy can induce durable remissions, but ~20% relapse early. The PI3K pathway is central to FL biology and multiple PI3K inhibitors (PI3Ki) are approved for FL, but the molecular profile of tumors most sensitive to PI3Ki is unknown. Further, PI3Ki are dosed indefinitely which contributes to toxicity and cost. Copanlisib is an IV inhibitor of PI3Kα and δ isoforms with high activity in relapsed FL. We hypothesized that patients with FL tumors most sensitive to PI3Ki will achieve deep and durable remissions after a fixed duration of copanlisib-based therapy. Here we report preliminary results of a response-adapted study using copanlisib and rituximab as frontline therapy for FL. Methods Pts with untreated grade 1-2, 3A FL, ≥stage 2, and any tumor burden are eligible if they meet criteria for need of systemic therapy that includes symptoms, increasing size of nodes, critical organ involvement, or impending organ compromise. No prior systemic therapy other than radiation is permitted. Frozen or archival tissue is required. Eligibility includes age ≥18 and adequate organ function unless involved by FL. Active HIV, CMV, Hep B or C, and autoimmune conditions requiring therapy are excluded. All pts receive PCP prophylaxis. Pts first receive copanlisib 60mg on days 1, 8, and 15 of a 28-day window to test activity of monotherapy. On-treatment tumor biopsies are optional after the window. Following the window, pts receive 6 cycles of copanlisib 60mg on days 1, 8, and 15 of a 28 day cycle along with rituximab 375mg weekly x 4 then on day 1 of each cycle. Streck tubes for circulating tumor DNA (ctDNA) are collected weekly during the window, after each cycle, and during surveillance. FDG-PET and CT scans are performed at baseline, after the window, and after cycles 3 and 6 to determine response. Patients with complete response (CR) after 6 cycles stop therapy. Patients with partial response (PR) after 6 cycles receive another 6 cycles of combination therapy with the copanlisib reduced to day 1 and 15 of each cycle. Non-responding patients will receive standard chemotherapy. The primary endpoint is the overall rate of CR with secondary endpoints of safety and duration of CR. Exploratory objectives include identification of a signature that predicts PI3Ki response. Results Ten pts have been enrolled and completed the copanlisib window. Median age was 50y (range, 28-77) including 3 (30%) over age 70. Seven (70%) pts had high-risk FLIPI scores ≥3 and two (20%) pts had Grade 3A FL. Comorbid conditions included prediabetes in 4 (40%) and hypertension in 4 (40%). All 10 (100%) pts had tumor reductions during copanlisib monotherapy with a median reduction of 41% (range 16-62%) (Figure 1). Four pts have completed 6 cycles of copanlisib and rituximab and all 4 (100%) have responded including 2 (50%) who achieved CR. In the two pts who achieved a PR after 6 cycles, one had a 90% tumor reduction and one had only persistent minimal residual disease in the bone marrow by flow cytometry. Toxicity was evaluated in 10 pts across 58 cycles and the most common have included rash (50%), diarrhea (50%) and mucositis (40%) which have all been G1 or G2 and successfully managed with supportive care and did not recur with subsequent cycles. One pt developed grade 3 neutropenia that responded to growth factors and did not recur. Four pts had dose delays due to rash (N=3) and lung infection (N=1). One pt had copanlisib reduced to 45mg due to recurrent rash and elevated liver tests. No pt has discontinued therapy. One pt required oral diabetic medications during therapy that were stopped after therapy completed. One pt had asymptomatic PCP pneumonia diagnosed during the copanlisib window prior to starting prophylaxis that was successfully treated while therapy continued. Conclusion Copanlisib is highly active in untreated FL and the first 10 (100%) patients all had tumor reductions after the first cycle of copanlisib monotherapy, including patients with high tumor bulk. Combination of copanlisib and rituximab can induce complete responses after only 6 cycles and without indefinite therapy. The safety profile includes rash and diarrhea that respond to supportive care and lessen on subsequent cycles. Updated clinical results from this ongoing trial (NCT03789240) along with the molecular profile of FL tumors will be presented at the meeting. Disclosures No relevant conflicts of interest to declare.

Abstract PO-46: Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma

Abstract Background: PI3Kδ is expressed in B cells and has a central role in the B-cell receptor signaling. Copanlisib is a highly selective PI3Kδ and PI3Kα inhibitor, and it is currently under clinical development in indolent lymphomas including marginal zone lymphoma (MZL). Copanlisib is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma. Nevertheless, a subset of patients can eventually relapse due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies; hence, we generated MZL cell lines resistant to copanlisib. Materials and Methods: Cells were kept on copanlisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 3 weeks of drug-free culture. Multidrug resistance phenotype was ruled out by confirming sensitivity to vincristine. Cells underwent transcriptome profiling (RNA-Seq) and immunophenotypic analysis. Results: The RES models were obtained from VL51 cell line with over 50-fold times higher IC50s than PAR counterparts. Of note, the copanlisib-resistant lines showed decreased sensitivity to other PI3K inhibitors such as duvelisib (50-fold) and idelalisib (5-fold) and to the BTK inhibitor ibrutinib (15-fold), suggesting that the mechanism observed here might drive resistance to other downstream B-cell receptor inhibitors. Gene expression profiles of RES showed the upregulation of cytokine signaling (IL1A, IL1B, CXCR4), NFkB (LTA, TNF), MAPK (RASGRP4, RASGRP2), and JAK-STAT (STAT3, JAK3) signaling pathways and negative regulators of apoptosis (CD44, JUN). Conversely, repressed genes in RES were involved in cell adhesion (ITGA4, ITGB1), antigen presentation (HLAs), and IFN response (PARP12, GBP6). Consistent with the overexpression of antiapoptotic signaling genes, RES cells exhibited also resistance to the BCL2-inhibitor venetoclax, either as a single as in combination with copanlisib. Flow cytometry confirmed the CXCR4 upregulation and the downregulation of CD49d (ITGA4), paired with reduced CD20 and CD81 surface expression. In accordance, addition of a CXCR4 inhibitor overcame resistance to copanlisib. Conclusions: We created a model of secondary resistance to the PI3K inhibitor copanlisib, derived from an MZL cell line. This model will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches. Indeed, we already identified novel potential targets, such as IL1 and CXCR4, that might be exploited in overcoming resistance to copanlisib and are worthy of further investigation. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Eugenio Gaudio, Roberta Bordone-Pittau, Marilia Barreca, Giulio Sartori, Chiara Tarantelli, Filippo Spriano, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Davide Rossi, Emanuele Zucca, Francesco Bertoni. Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-46.

Combining Radiotherapy with PI3K Isoform-Selectivity Inhibitor Increases Radiosensitivity And Suppresses Tumor Growth In Glioblastoma Cells

Glioblastoma (GBM) is the most malignant cancer that occurs within the brain and shows the dismal prognosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway plays a critical role in GBM. Also, the activation of Akt due to irradiation induces radioresistance. But, the pan-PI3K inhibitors have side-effects in clinical trial. Different PI3K isoforms play non-redundant roles in particular tumor. So, it is expected that selective inhibition of PI3K isoforms decrease side effects. Therefore, we investigated whether combining radiotherapy with the PI3K isoform inhibitor decreases radioresistance and tumor growth in GBM. Glioma 261 expressed luciferase (GL261-luc) cell lines were used to identify the effects of combination therapy. GL261-luc were irradiated 1Gy with or without PI3K isoform inhibitors. After irradiation, clonogenic assay was performed to confirm the GBM survival rate and observed inhibition of PI3K-AKT signaling pathway using western blot (WB) assay. In order to confirm to repair DNA double strand breaks (DSB), the expression of phospho-H2A.X was confirmed by immunofluorescence. Also, to observe the effects of combination therapy in GBM in vivo, an orthotopic mouse model was established by intracranial injection implanting GL261-luc. Irradiation was performed specifically to brain with 2Gy, 3Gy and 4Gy for 5 days with or without PI3K-α isoform inhibitor. We observed tumor growth using IVIS image and analyzed splenocyte RNA and protein level using RT-PCR and WB. According to inhibition of PI3K-AKT signaling, GL261-luc radiosensitivity was significantly increased. GL261-luc irradiated by 1Gy was suppressed cell growth about 70% compared to control (p<0.001). When irradiated with PI3K-isform inhibitor, each survival rate was about 18% (PI3K-pan, p<0.00001), 25% (PI3K-α, p<0.00001), 30% (PI3K-δ, p<0.00001), 45% (PI3K-γ/δ, p<0.00001). Also, the expression of phospho-H2A.X, DNA DSB marker, was increased by combination therapy. Also, GL261-luc irradiated with PI3K- isoform inhibitor was decreased the ability of migration. Especially, the effects of combining radiotherapy with PI3K-α inhibitor was similar as much as with PI3K-pan inhibitor (In vitro). So, we selected PI3K-α inhibitor and analyzed combination therapy effects in GBM in vivo. We demonstrated that combining radiotherapy with the PI3K-α isoform inhibitor was markedly delayed tumor growth than without inhibitor (p<0.0001). In addition, the expression of PD1, regulator of cancer immune system, in splenocyte was increased by combination therapy (p<0.001) (In vivo). Our findings demonstrate that combining radiotherapy with the PI3K-α isoform increased radiosensitivity. Also, GBM tumor growth was markedly suppressed and increased immunogenic marker such as PD1. Combining radiotherapy with the PI3K-α isoform inhibitor would be a potential strategy for overcoming radioresistance in GBM, but further study should be necessary.

Identification of methyl (5-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-4-morpholinopyrrolo[2,1-f][1,2,4]triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)carbamate (CYH33) as an orally bioavailable, highly potent, PI3K alpha inhibitor for the treatment of advanced solid tumors.

In various human cancers, PI3Ks pathway is ubiquitously dysregulated and thus become a promising anti-cancer target. To discover new potent and selective PI3K inhibitors as potential anticancer drugs, new pyrrolo[2,1-f][1,2,4]triazines were designed, leading to the discovery of compound 37 (CYH33), a selective PI3Kα inhibitor (IC50=5.9nM, β/α, δ/α,γ/α=101-, 13-, 38-fold). Western blot analysis confirmed that compound 37 could inhibit phosphorylation of AKT in human cancer cells to modulate the cellular PI3K/AKT/mTOR pathway. And further evaluation invivo against SKOV-3 xenograft models demonstrated that a dose-dependent antitumor efficacy was achieved.

Cardiovascular toxicity of PI3Kα inhibitors.

The phosphoinositide 3-kinases (PI3Ks) are a family of intracellular lipid kinases that phosphorylate the 3'-hydroxyl group of inositol membrane lipids, resulting in the production of phosphatidylinositol 3,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate. This results in downstream effects, including cell growth, proliferation, and migration. The heart expresses three PI3K class I enzyme isoforms (α, β, and γ), and these enzymes play a role in cardiac cellular survival, myocardial hypertrophy, myocardial contractility, excitation, and mechanotransduction. The PI3K pathway is associated with various disease processes but is particularly important to human cancers since many gain-of-function mutations in this pathway occur in various cancers. Despite the development, testing, and regulatory approval of PI3K inhibitors in recent years, there are still significant challenges when creating and utilizing these drugs, including concerns of adverse effects on the heart. There is a growing body of evidence from preclinical studies revealing that PI3Ks play a crucial cardioprotective role, and thus inhibition of this pathway could lead to cardiac dysfunction, electrical remodeling, vascular damage, and ultimately, cardiovascular disease. This review will focus on PI3Kα, including the mechanisms underlying the adverse cardiovascular effects resulting from PI3Kα inhibition and the potential clinical implications of treating patients with these drugs, such as increased arrhythmia burden, biventricular cardiac dysfunction, and impaired recovery from cardiotoxicity. Recommendations for future directions for preclinical and clinical work are made, highlighting the possible role of PI3Kα inhibition in the progression of cancer-related cachexia and female sex and pre-existing comorbidities as independent risk factors for cardiac abnormalities after cancer treatment.

Co-Occurring Gain-of-Function Mutations in &lt;i&gt;HER2&lt;/i&gt; and &lt;i&gt;HER3&lt;/i&gt; Modulate HER2/HER3 Activation, Breast Cancer Progression, and HER2 Inhibitor Sensitivity

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrated computational structural modeling with biochemical and cell biological analyses. Computational modeling predicted that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3, stabilizes the HER2 active conformation, and reduces binding of HER2 to its inhibitor neratinib, potentially explaining the lower clinical response of HER2/HER3 co-mutant tumors. Co-expression of mutant HER2 and HER3 enhanced HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which was reversed by treatment with PI3Kα inhibitors.SignificanceThe HER2 TKI neratinib has shown clinical activity against cancers harboring HER2 activating mutations. However, not all HER2-mutant tumors respond, suggesting that co-occurring mutations may limit sensitivity. We found that co-occurring HER3 mutations resulted in reduced sensitivity to HER2 TKIs and HER2 antibodies by disrupting neratinib binding and preventing the dissociation of HER2/HER3, respectively. Cells expressing HER2/HER3 mutations were sensitive to the combination of a HER2 TKI and a PI3Kα inhibitor, suggesting a potential therapeutic strategy against these refractory tumors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a lesser response to neratinib in HER2-mutant cancers.

CDK 4/6 Inhibition Overcomes Acquired and Inherent Resistance to PI3Kα Inhibition in Pre-Clinical Models of Head and Neck Squamous Cell Carcinoma.

Activating alterations in PIK3CA, the gene coding for the catalytic subunit of phosphoinositide-3-kinase (PI3K), are prevalent in head and neck squamous cell carcinoma (HNSCC) and thought to be one of the main drivers of these tumors. However, early clinical trials on PI3K inhibitors (PI3Ki) have been disappointing due to the limited durability of the activity of these drugs. To investigate the resistance mechanisms to PI3Ki and attempt to overcome them, we conducted a molecular-based study using both HNSCC cell lines and patient-derived xenografts (PDXs). We sought to simulate and dissect the molecular pathways that come into play in PIK3CA-altered HNSCC treated with isoform-specific PI3Ki (BYL719, GDC0032). In vitro assays of cell viability and protein expression indicate that activation of the mTOR and cyclin D1 pathways is associated with resistance to PI3Ki. Specifically, in BYL719-resistant cells, BYL719 treatment did not induce pS6 and pRB inhibition as detected in BYL719-sensitive cells. By combining PI3Ki with either mammalian target of rapamycin complex 1 (mTORC1) or cyclin D1 kinase (CDK) 4/6 specific inhibitors (RAD001 and abemaciclib, respectively), we were able to overcome the acquired resistance. Furthermore, we found that PI3Ki and CDK 4/6 inhibitors have a synergistic anti-tumor effect when combined in human papillomavirus (HPV)-negative/PIK3CA-WT tumors. These findings provide a rationale for combining PI3Ki and CDK 4/6 inhibitors to enhance anti-tumor efficacy in HNSCC patients.

Abstract B06: Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer

Abstract Activating KRAS mutation is the most common driver mutation in the initiation and promotion of non-small cell lung cancer (NSCLC). Direct inhibition of the enzymatic activity of mutated Kras has been proved unsuccessful. Deregulation of PI3K-Akt-mTOR signaling, one of the most important downstream effectors of Kras, plays important roles in tumorigenesis and drug resistance in NSCLC. In this study, we found that a novel PI3Kα-specific inhibitor, CYH33, possessed variable activity against a panel of Kras-mutated NSCLC cell lines. Though CYH33 blocked Akt phosphorylation in all tested cells, phosphorylated Rb decreased in CYH33-sensitive but not resistant cells, which was consistent with G1 phase arrest in CYH33-sensitive cells. Consequently, combination of a CDK4/6 inhibitor PD0332991 with CYH33 displayed synergistic activity against the proliferation of both CYH33-sensitive and -resistant cells, which was accompanied with enhanced cell population in G1 phase compared to single-agent treatment. Moreover, downregulation of cyclin D1 with siRNA sensitized A549 and H23 cells to CYH33. Reciprocally, CYH33 abrogated PD0332991-induced upregulation of phosphorylated Akt and cyclin D1 in A549 cells. Cotreatment of these two drugs demonstrated synergistic activity against A549 and H23 xenografts with enhanced inhibition on the phosphorylation of Akt and Rb compared to administration of CYH33 or PD03332991 alone. Thus, simultaneous inhibition of PI3Kα and CDK4/6 displayed synergistic activity against Kras-mutated NSCLC and provided a mechanistic rationale for a combination approach using PI3Kα inhibitor and CDK4/6 inhibitor CYH33 for the therapy of Kras-mutated NSCLC. Citation Format: Yu-xiang Wang, Xian Li, Xue-ling Liu, Bo-bo Wang, Yi Chen, Chun-hao Yang, Jian Ding, Ling-hua Meng. Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B06.

Abstract A25: Activating alterations of p110 subunits determine PI3K isoform selectivity in prostate cancer

Abstract Oncogenic activity of PI3K mutated tumors is often dependent on different PI3K isoforms. In breast cancer PI3Kα activating mutations are dominant drivers, whereas in PTEN-deficient prostate cancer such activity is thought to be driven by PI3Kβ. In particular, loss of PTEN function is seen in 40% of metastatic prostate cancer patients, which remains a challenging clinical problem. Recent success of selectively targeting PI3Kα or PI3Kβ to treat disease caused by hyperactivation of PI3K pathway sheds light on further developing such agents to a boarder disease spectrum. Yet relatively little is known about how different PI3K isoforms are selected as dominant drivers in different genetic and pathologic contexts. Here we use multiple prostate cancer cell lines and organoid models to study the mechanisms of PI3K isoform dependency in prostate cancer. We found PI3K isoform dependency is not always correlated with PTEN status across a panel of prostate cancer models in which different levels of PI3K signaling are observed. However, such dependency can be shifted towards either PI3Kα or PI3Kβ in the presence of its corresponding activating alteration, regardless of its original isoform dependency. Interestingly, such PI3K isoform activating alteration is able to further enhance PI3K signal in the setting of PTEN loss. PI3Kα mutant models are more sensitive to the α selective inhibitor BYL719 while PI3Kβ mutant models are more sensitive to the β selective inhibitor AZD8186. Besides, we found that PI3Kα mutant prostate tumors are sensitive to PI3Ksy inhibition by AZD8186 at a high dosage both in vitro and in vivo. High dose of AZD8186 also inhibits PI3K signaling in PI3Kα mutant breast cancer models. These data raise the possibility that the PI3Kβ selective inhibitor AZD8186 can also target PI3Kα at a high dose. In an ongoing clinical trial at MSK, a prostate cancer patient with PI3Kα mutation also responded to PI3Kβ inhibition. This finding reveals a new mechanism of how PI3K activating mutations determine PI3K isoform selectivity regardless of PTEN status. Results from this study also present new opportunities to treat cancer patients with PI3Kα or PI3Kβ inhibition. Citation Format: Zeda Zhang, Ninghui Mao, Young Sun Lee, Dan Li, Neal Rosen, Charles Sawyers, Brett Carver. Activating alterations of p110 subunits determine PI3K isoform selectivity in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A25.

Abstract A21: Compound PIK3CA mutations support a mutational dose-response model for oncogene activation and response to PI3K inhibitor targeted therapy in breast cancer

Abstract PIK3CA mutations represent the most frequent oncogenic driver lesion found across all human cancers. Given that single PIK3CA mutations are a predictive biomarker for response to PI3K inhibition, and that these clinical benefits are small, we hypothesized that additional genomic factors may cooperate with single PIK3CA mutations in oncogenesis and response to PI3K inhibitors. We analyzed several large pan-cancer datasets and have discovered a phenomenon of dual PIK3CA mutations in 15% of all PIK3CA-mutant cancers, including breast cancer. Dual PIK3CA mutations are clonal, are found in both primary untreated and metastatic tumors, and are enriched at conserved amino acid positions. We have proved that the majority of dual PIK3CA mutations—including the most frequent dual PIK3CA mutant combinations in breast cancer—are in cis compound mutations (i.e., on the same allele, resulting in a protein molecule with two mutations) in patient samples and cell lines, as shown through multiple sequencing techniques including long-range single-molecule real-time sequencing (SMRT-seq). Compound PIK3CA mutations increase PI3K pathway signaling and growth proliferation in nontransformed cells and cancer cells in vitro and in vivo. Biochemical experiments using recombinant PI3K complexes reveal a mechanism where compound mutants increase PI3K complex thermal instability, resulting in increased kinase activity and liposome binding as compared to single mutants. Compound PIK3CA mutations render cells more sensitive to the PI3Kα inhibitors BYL719 and GDC-0077 than single PIK3CA mutations. These data translate to the clinic, where patients with dual PIK3CA-mutant metastatic breast cancer exhibit more durable responses to PI3K inhibition versus patients with single PIK3CA-mutant breast tumors. Our data support a novel mutational dose-response model for oncogene activation, where compound mutations amplify PI3K signaling and growth to a greater degree than single mutants, resulting in increased sensitivity to PI3Kα inhibitor targeted therapy. These findings also have therapeutic relevance, where compound PIK3CA mutations may be a superior predictive biomarker for response to PI3Kα inhibitors than single PIK3CA mutations, across PIK3CA-mutant cancers. Citation Format: Neil Vasan, Jared Johnson, Hong Shao, Pedram Razavi, Alexander Gorelick, Erik Ladewig, Alesia Antoine, Hardik Shah, Eneda Toska, Guotai Xu, Abiha Kazmi, Barry Taylor, Komal Jhaveri, Maura Dickler, Elisa de Stanchina, Eduard Reznik, Raul Rabadan, Melissa Smith, Robert Sebra, Lewis Cantley, Maurizio Scaltriti, Jose Baselga. Compound PIK3CA mutations support a mutational dose-response model for oncogene activation and response to PI3K inhibitor targeted therapy in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A21.

Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER+ Breast Cancer

SummaryPIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER+ breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[18F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance.

PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition

PIK3CA hotspot mutation is well established as an oncogenic driver event in cancer and its durable and efficacious inhibition is a focus in the development and testing of clinical cancer therapeutics. However, hundreds of cancer-associated PIK3CA mutations remain uncharacterized, their sensitivity to PI3K inhibitors unknown. Here, we describe a series of PIK3CA C-terminal mutations, primarily nucleotide insertions, that produce a frame-shifted protein product with an extended C terminus. We report that these mutations occur at a low frequency across multiple cancer subtypes, including breast, and are sufficient to drive oncogenic transformation in vitro and in vivo. We demonstrate that the oncogenicity of these mutant p110α proteins is dependent on p85 but not Ras association. P110α-selective pharmacologic inhibition blocks transformation in cells and mammary tumors characterized by PIK3CA C-terminal mutation. Taken together, these results suggest patients with breast and other tumors characterized by PIK3CA C-terminal frameshift mutations may derive benefit from p110α-selective inhibitors, including the recently FDA-approved alpelisib.

Discovery and Development of SPR519 as a Potent, Selective, and Orally Bioavailable Inhibitor of PI3Kα and mTOR Kinases for the Treatment of Solid Tumors.

Herein, we report the identification and preclinical profile of a lead compound 10, (SPR519) as an equally potent dual inhibitor of PI3Kα and mTOR kinases. SPR519 exhibits an EC50 of low sub-micromolar range among various tested cancer cell lines such as A2780 (0.23 μM), PC3 (0.48 μM), and SKOV3 (0.50 μM). When administrated orally, it shows a considerably high plasma exposure (area under curve: 26,858 nM/h at 1 mg/kg) in mice. Moreover, it is found to be safe in animals with a dose of 30 mg/kg BID for 12 days in the dose tolerance study. SPR519 did not show any CYP or hERG liability. The identified lead compound demonstrates significant efficacy and bioavailability in ovarian and colon cancer xenograft models when evaluated for dose-ranging efficacy studies, at a dose as low as 2.5 mg/kg.

1933MO TransFAL: Establishment of clinical trial-matched luminal breast cancer patient-derived xenografts (PDX) for translational studies

PARSIFAL was a randomized phase II trial aimed at evaluating the efficacy of palbociclib (P) plus either fulvestrant (F) or letrozole (L) in Estrogen Receptor[+]/Human Epidermal Growth Factor Receptor 2[-] metastatic breast cancer. The present study focused on establishing luminal breast cancer PDX models from a cohort of enrolled patients in an effort to develop preclinical models that recapitulate the disease before and after treatment with P and endocrine therapy (ET). 14 pre- and 18 post-treatment tumors were transplanted into immunocompromised mice. Xenografts were profiled using a capture-based exome sequencing panel and analyzed by immunohistochemistry as well as for the in vivo antiproliferative activity of P plus the patient-matched ET. Models of acquired resistance were generated from responder models and characterized. Predictive markers of response were analyzed and, in the case of non-responder models, additional treatments were tested in vivo and/or ex vivo. Successful engraftments were established in 3 of the 32 tumors (take rate 9%, 95% CI 1.8% -23.1%). Of those, PDX426 and PDX446 derived from pre-treatment tumors of patients receiving P+L and P+F, respectively, and PDX450 from a post-treatment tumor of a patient treated with P+L. PDX426 harbored an FGFR1 amplification and PDX446 harbored concomitant PIK3CA and PTEN mutations. Both PDXs expressed normal levels of pRb and cyclin E1. PDX446 showed sensitivity to P+F or P+L for >95 days, resembling the patient’s clinical response. Of note, PDX446 was also sensitive to single-agent F or L. The model of acquired resistance to P+F exhibited lower expression of pRb than the sensitive model. PDX450 harbored concomitant ESR1, PIK3CA, and PTEN mutations and expressed low pRb. Despite an initial response, all tumors progressed to P+L after 80 days, matching the clinical disease progression. The P+L-resistant tumors were also resistant to P+F in vivo but sensitive to the PI3K-α inhibitor alpelisib plus F ex vivo. PDX models showed comparable treatment responses to those observed clinically, providing information about the additional benefit of P to ET alone as well as effective treatment options in the post-P setting.

LBA18 Overall survival (os) results from SOLAR-1, a phase III study of alpelisib (ALP) + fulvestrant (FUL) for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC)

Phosphatidylinositol 3-kinase (PI3K) pathway hyperactivation due to PIK3CA mutations contributes to poor survival in patients (pts) with HR+, HER2– ABC (Mosele 2020). In the phase III SOLAR-1 trial of pts with progression on/after aromatase inhibitor (AI), the PI3Kα inhibitor ALP together with FUL significantly improved progression-free survival (PFS) in the PIK3CA-mutant (mut) cohort (HR 0.65; 95% CI 0.50-0.85; P<0.001; median [m] PFS 11.0 mo with ALP + FUL vs 5.7 mo with placebo [PBO] + FUL), a population with poor prognosis. At final PFS analysis, the first interim OS results were immature. Men/postmenopausal women (N=572) with HR+, HER2– ABC with no prior chemotherapy treatment (tx) and whose disease progressed on/after AI were randomized 1:1 to receive ALP (300 mg PO QD) or PBO, + FUL (500 mg IM on D1 and D15 of C1 then D1 of each 28-d cycle) and stratified by presence of lung and/or liver metastases and prior cyclin-dependent kinase 4/6 inhibitor. OS in the PIK3CA-mut cohort, a key secondary endpoint, was evaluated by Kaplan-Meier methodology; a 1-sided stratified log-rank test was performed with an O’Brien-Fleming efficacy boundary of P≤0.0161. With mOS follow-up of 30.8 mo, mOS was 39.3 mo (95% CI 34.1-44.9) with ALP + FUL and 31.4 mo (95% CI 26.8-41.3) with PBO + FUL (HR 0.86; 95% CI 0.64-1.15; P=0.15); OS did not cross the prespecified boundary. Median time to chemotherapy (TTC) was 23.3 mo (95% CI 15.2-28.4) with ALP + FUL and 14.8 mo (95% CI 10.5-22.6) with PBO + FUL (HR 0.72; 95% CI 0.54-0.95). In pts with lung and/or liver metastases, mOS was 37.2 mo (95% CI 28.7-43.6) with ALP + FUL and 22.8 mo (95% 19.0-26.8) with PBO + FUL (HR 0.68; 95% CI 0.46-1.00). No new safety signals were observed with longer follow-up. Though not statistically significant, OS was prolonged by a clinically relevant ≈8 mo with ALP when added to FUL in HR+, HER2−, PIK3CA-mut ABC. mTTC was also prolonged with ALP + FUL vs PBO + FUL. Coupled with the statistically and clinically significant PFS, these data further support ALP + FUL in this poorer prognostic population of pts with PIK3CA-mut ABC.

The PI3Kα inhibitor DFX24 suppresses tumor growth and metastasis in non-small cell lung cancer via ERK inhibition and EPHB6 reactivation

EPHB6 is a metastasis inhibitory gene that is frequently decreased or deficiency in non-small cell lung cancer (NSCLC), which contributed to the subsequent development of distant metastasis. These suggested the possibility that reactivation of EPHB6 might prevent the metastasis of NSCLC. Nevertheless, EPHB6 expression might also promote cancer cell growth and inhibit cell apoptosis by activating Akt and ERK pathway, apart from inhibition of migration and invasion. In the present study, we developed a novel quinazolin-4(3H)-one analog (DFX24) as a potential PI3Kα inhibitor, which inhibited both cell proliferation and metastasis of NSCLC cell lines. Investigation to the molecular mechanisms revealed DFX24 inhibited the cell growth and metastasis via inhibition of PI3Kα and ERK activity, as well as the increase in EPHB6 expression. In addition, DFX24 also induced cell cycle arrest and tumor cell apoptosis by inhibiting PI3K/Akt pathway and activating mitochondria-dependent pathway, respectively. These findings suggested that DFX24 might be considered as a novel drug candidate and may provide a potential therapy for NSCLC.