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Metabolite changes that constitute the Venn diagram in Figure 2. Significant changes listed are reductions (labelled "-1") or increases (labelled "1") in concentrations relative to the relevant controls.

Summary of baseline reproducibility of candidate plasma metabolite biomarkers (n=17 patients).

Characteristics of dose-dependent changes in candidate plasma metabolite pharmacodynamic biomarker concentrations in 41 patients, as determined on day 1 of pictilisib clinical trial. Cells with p-values

Changes in candidate plasma metabolite pharmacodynamic biomarker concentrations over first 2 weeks in 17 patients treated with {greater than or equal to}330 mg once-daily of pictilisib. Cells with p-values

Heat map of changes (relative to vehicle control) in candidate plasma and tumor metabolite biomarkers at 8 hours post-dosing of mice bearing U87MG xenografts with pictilisib 75mg/kg or buparlisib 60mg/kg (a, acyl; aa, acyl-acyl; ae, acyl-alkyl; Cx:y, where x is the number of carbons in the fatty acid side chain; y is the number of double bonds in the fatty acid side chain; AC, acylcarnitine; DC, decarboxyl; M, methyl; OH, hydroxyl; PC, phosphatidylcholine)

Changes in plasma metabolites across timepoints by patient/sample.

Purpose: Oncogenic signaling in gastrointestinal stromal tumors (GIST) is sustained via PI3K/AKT pathway. We used a panel of six GIST xenograft models to assess efficacy of GDC-0941 as single agent or in combination with imatinib (IMA).Experimental Design: Nude mice (n = 136) were grafted bilaterally with human GIST carrying diverse KIT mutations. Mice were orally dosed over four weeks, grouped as follows: (A) control; (B) GDC-0941; (C) imatinib, and (D) GDC+IMA treatments. Xenografts regrowth after treatment discontinuation was assessed in groups C and D for an additional four weeks. Tumor response was assessed by volume measurements, micro-PET imaging, histopathology, and immunoblotting. Moreover, genomic alterations in PTEN/PI3K/AKT pathway were evaluated.Results: In all models, GDC-0941 caused tumor growth stabilization, inhibiting tumor cell proliferation, but did not induce apoptosis. Under GDC+IMA, profound tumor regression, superior to either treatment alone, was observed. This effect was associated with the best histologic response, a nearly complete proliferation arrest and increased apoptosis. Tumor regrowth assays confirmed superior activity of GDC+IMA over imatinib; in three of six models, tumor volume remained reduced and stable even after treatment discontinuation. A positive correlation between response to GDC+IMA and PTEN loss, both on gene and protein levels, was found.Conclusion: GDC+IMA has significant antitumor efficacy in GIST xenografts, inducing more substantial tumor regression, apoptosis, and durable effects than imatinib. Notably, after treatment withdrawal, tumor regression was sustained in tumors exposed to GDC+IMA, which was not observed under imatinib. Assessment of PTEN status may represent a useful predictive biomarker for patient selection. Clin Cancer Res; 19(3); 620–30. ©2012 AACR.

PDF file - 566K, Xenograft tumor volume assessment, combined for all models. Tumor volume is expressed in percentage, as ratio between time point x baseline. Data are presented for all models (A), based on PTEN protein expression (B), and PTEN status by FISH (C). In GIST-PSW GDC-0941 treatment as single agent lasted 19 days (*). The dotted line shows the time of treatment withdrawal.

PDF file - 1222K, PTEN protein expression. Strong expression of PTEN was observed in GIST-DFR (A), GIST-BOE (B), GIST882 (C) and GIST48 (D). In GIST-PSW (E) and GIST882Ly (F) the majority of the tumor cells were PTEN negative, only scattered cells showed weak cytoplasmatic staining. The endothelium of blood vessels was used as internal positive control.

PDF file - 58K, Xenografts used in GDC-0941 efficacy experiments. All mice were subcutaneously transplanted with tumor fragment on both sides.

PDF file - 4757K, Western blotting showing KIT oncogenic signaling cascade. Three different tumor lysates were analyzed per treatment group.

PDF file - 1683K, Representative micro-PET images from GIST-PSW (A) and GIST-DFR (B) mice treated with IMA or GDC+IMA are shown. Scans were perfromed at baseline, week four (end of the treatment) and weeks six and eight - during treatment discontinuation.

PDF file - 2344K, Representative images of control tumors from GIST882Ly (A-E) and GIST-DFR (F-J). The H&E staining was shown to illustrate tumor morphology (A, F). Expression of KIT (B, G), ANO1 (C, H), ETV1 (D, I) and CD44 (E, J) were studied to further characterize GIST xenograft models.

PDF file - 84K, Xenograft models characterization.

PDF file - 240K

[Image: see text] Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms—as a full antagonist and selective estrogen receptor degrader (SERD)—but lacks oral bioavailability. Thus, we envisioned a “best-in-class” molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

The phosphatidylinositol 3kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Hotspot mutations of PIK3CA are common in all subtypes of breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent ATP-competitive inhibitor of PI3K alpha (IC50 = 0.038 ± 0.003 nM), thereby inhibiting the phosphorylation of membranebound PIP2 to PIP3. Biochemically, GDC-0077 is more than 300fold selective over the other Class I PI3K isoforms, 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. Mechanism of action studies indicate that GDC-0077 treatment leads to degradation of the mutant PI3K alpha protein in a proteasome dependent fashion, without changing WT PI3K alpha levels. Drug treatment results in sustained reduction of PI3K pathway biomarkers pAkt and pPRAS40, inhibition of cell proliferation, and increased apoptosis in human PIK3CA mutant breast cancer cell lines, to a greater extent than other non-degrading inhibitors such as alpelisib. Combination studies of GDC-0077 with the CDK4/6 inhibitor palbociclib demonstrate a stronger effect in estrogen-depleted HR+ PIK3CA mutant breast cell lines, compared to cells grown in the presence of estrogen. Daily dosing of GDC-0077 in PIK3CA mutant breast cancer patient derived xenograft (PDX) models (n=4) resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo combination efficacy studies of GDC-0077 with fulvestrant and palbociclib also indicate the triple combination results in the greatest efficacy. Collectively, our preclinical data support evaluation of GDC-0077 in a triple combination with endocrine therapy and CDK4/6 inhibition in the clinic, in patients with locally advanced or metastatic hormone receptor positive breast cancer whose tumors harbor mutant PIK3CA. Citation Format: Kyle Edgar, Rebecca Hong, Kyung Song, Steven Schmidt, Marc Hafner, Alfonso Arrazate, Erin Williams, Cecile De La Cruz, Jason Oeh, Deepak Sampath, Steve Staben, Lori Friedman. GDC-0077 is a selective PI3K alpha inhibitor with robust efficacy in PIK3CA mutant hormone-positive breast cancer models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-23.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Seeing double can be a good thingMany human breast cancers harbor activating mutations inPIK3CA, the gene coding for the catalytic subunit of phosphoinositide 3-kinase (PI3K). Clinical trials are underway to evaluate the efficacy of PI3K inhibitors in cancer patients. Vasanet al.found unexpectedly that a subset of breast cancers harbor not one—but two—PIK3CAmutations, and the mutations occur on the same allele (see the Perspective by Toker). In model systems, the double mutations hyperactivate PI3K signaling and enhance tumor growth. Preliminary analysis of clinical trial data suggests that breast cancers with double mutations are more responsive to PI3K inhibitors than those with a single mutation.PIK3CAmutational status could help identify the breast cancer patients most likely to benefit from these drugs.Science, this issue p.714; see also p.685

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. Significance: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors. See related commentary by Vanhaesebroeck et al., p. 20. This article is highlighted in the In This Issue feature, p. 1

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.

It has been 40 years since the US Food and Drug Administration approved the estrogen receptor (ER) antagonist tamoxifen for the treatment of ER-positive breast cancer, ushering in the era of targeted therapy coupled with a companion diagnostic. The prostate cancer field quickly followed suit with the approval of the androgen receptor (AR) antagonist bicalutamide. In the years since, there has been sustained scientific interest in understanding these hormone-dependent signaling pathways and in drug discovery efforts to identify novel hormone-directed therapeutic agents. Recently, there have been breakthrough discoveries relating to mechanisms that enable reactivation of ER and AR signaling in the presence of antihormonal agents and that enable loss of hormone dependency, providing multiple routes of acquired resistance to hormone therapy. This review discusses parallels between breast and prostate cancer, including their pathobiologies, existing therapeutic modalities, acquired resistance to such therapeutics, and novel therapies being developed to target distinct states of resistance.

Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER+ BC. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. ER antagonists that are efficacious against ligand-dependent and ligand-independent, constitutively active ESR1 mutant tumors may be of substantial therapeutic benefit. GDC-0927 (formerly known as SRN-927) is a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist/ER degrader (SERD) that induces tumor regression in ER+ BC patient-derived xenograft models. Methods: A phase I dose escalation study with 3+3 design was conductedin postmenopausal women with ER+ (HER2-) metastatic BC (progressing ≥ 6 months on endocrine therapy and with ≤ 2 prior chemotherapies in the advanced or metastatic setting) to determine the safety, pharmacokinetics (PK) and the recommended Phase 2 dose (RP2D) of GDC-0927. Pharmacodynamic (PD) activity was assessed with [18F]-fluoroestradiol (FES)-PET scans. Plasma PK samples (after single dose and at steady state), CT scans, and when feasible, pre and on-study tumor biopsies were obtained Results: From March 16, 2015 to March 17, 2017 patients (pts) with a median age of 53 years (range 44-69) and a median number of prior therapies for MBC 4 (range 1-7) were enrolled at 3 total daily dose levels (600, 1000, 1400 mg) once daily (QD) given orally with fasting (n = 12). Increases in GDC-0927 exposure were approximately dose proportional. Treatment related adverse events (AEs) were all grade 1 or 2. The most common treatment-related AEs were nausea (54%, n = 7), diarrhea (46%, n = 6), elevated aspartate aminotransferase (39%, n = 5) and anemia, constipation, (each 31%, n = 4). Treatment interruption was required for 2 pts due to nausea and vomiting. Of those pts with FES-PET avid disease at baseline (9 of 12), all post-therapy scans showed complete or near complete (> 90%) suppression of FES uptake to background levels, including pts with ESR1 mutations. Evidence of reduced ER levels and Ki67 staining was observed in on-treatment biopsies. Five of 12 pts (1 at 600 mg and 4 at 1400 mg) were on study ≥ 24 weeks (CBR = 41.6 %) with the best overall response of stable disease with 1 patient (ESR1 mt+ D538G) on study for over 490 days. There were no dose limiting toxicities and no SAEs related to study drug. R2PD was 1400 mg and was selected for single arm dose-expansion which is now complete with last patient enrolled on March 17, 2017. Updated results from dose-escalation and dose-expansion will be presented at the meeting (N = 43). Conclusions: GDC-0927 appears well-tolerated to date with PK exposure supporting QD dosing, evidence of robust PD target engagement, and encouraging anti-tumor activity in heavily pretreated pts with advanced or metastatic ER+ BC, including pts with ESR1 mutations. Citation Format: Dickler MN, Villanueva R, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez-Martin A, Cortes J, Martin M, Giltnane J, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Spoerke JM, Metcalfe C, Liu L, Li R, Morley R, McCurry U, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Bardia A. A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader (SERD), GDC-0927, in postmenopausal women with estrogen receptor positive (ER+) HER2-negative metastatic breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-10.

The phosphatidylinositol 3 kinase (PI3K)/AKT/ mechanistic 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 different mechanisms has been described in solid tumor malignancies, including activating and transforming mutations and amplification of PIK3CA, that encodes the p110alpha subunit of PI3K. Indeed, PIK3CA hotspot mutations are highly prevalent in breast cancer, occurring in approximately 40% of HR+ tumors. The clinical candidate GDC-0077 is a potent inhibitor of PI3Kalpha (IC50 = 0.038 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 >300-fold more selective for PI3Kalpha over the other class I PI3K isoforms (beta, delta, and gamma) and >2000-fold more selective over PIK family members. Furthermore, GDC-0077 is more selective for mutant versus wild-type PI3Kalpha in cell based assays. The improved biochemical selectivity of GDC-0077 relative to PI3Kdelta translated in human CD69+ B-cells, which are primarily dependent on PI3Kdelta for proliferation and survival, and were more sensitive (based on reduction of cell number) to the PI3Kalpha/delta selective inhibitor taselisib (GDC-0032) than to GDC-0077. Mechanism of action (MOA) studies indicate that GDC-0077 selectively degrades mutant PI3Kalpha in a proteasome-dependent fashion resulting in reduction of PI3K pathway activity 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, oral daily treatment of patient-derived PIK3CA-mutant breast cancer xenograft models with GDC-0077 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 therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or a CDK4/6 inhibitor (palbociclib). Collectively, preclinical studies provide rationale for evaluating GDC-0077, a PI3Kalpha selective inhibitor that degrades mutant p110alpha protein, as a single agent and in combination with endocrine and targeted therapies that may provide additional benefit to patients with locally advanced or metastatic hormone receptor+ breast cancers that harbor PIK3CA mutations. Citation Format: Hong R, Edgar K, Song K, Steven S, Young A, Hamilton P, Arrazate A, De La Cruz C, Chan C, Pang J, Salphati L, Belvin M, Nannini M, Staben S, Friedman L, Sampath D. GDC-0077 is a selective PI3Kalpha inhibitor that demonstrates robust efficacy in PIK3CA mutant breast cancer models as a single agent and in combination with standard of care therapies [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-14.

Introduction: Adenosine is an anti-inflammatory and immunosuppressive metabolite, that signals to diminish activation and proliferation of cytotoxic T-cells, impair activity of natural killer cells and CD4 + effector T-cells, and promote the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, is required to convert AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many tumors and correlates with unfavorable clinical outcome. Bone marrow (BM) aspirates from multiple myeloma (MM) patients have shown increased adenosine levels correspond with disease progression [Horenstein et al. Mol Med. 2016,22:694-704] In addition to the adenosine rich feature of MM, multiple cell types within the MM BM niche express the enzymes required for adenosine production from both NAD and ATP precursors, including CD38, CD203a, CD39 and CD73. Previously, we demonstrated that dysfunctional plasmacytoid dendritic cells (pDCs) predominantly found in the BM of MM patients contribute to MM cell growth, survival, and suppression of antitumor immunity [Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444]. We recently discovered that the interaction between pDCs and MM cells increased CD73 transcript and protein levels in both cell types, implicating a role for adenosine signaling via CD73 signaling axis in MM. Together, these MM disease features indicate that reducing the level of adenosine via inhibition of CD73 may represent a unique vulnerability and treatment strategy for MM. Methods: To understand the functional consequence of CD73 inhibition in MM, autologous ex vivo cell assays using freshly isolated BM aspirates from MM patients were used to detect changes in immune cell function and MM cell viability upon treatment with OP-5558, a potent and selective CD73 small molecule inhibitor which is an analog of the clinical candidate, ORIC-533. The majority of BM samples utilized were from patients with relapsed or refractory MM after at least three lines of therapy including immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, as well as a patient with relapsed MM post BCMA-CAR-T therapy. Results: In BM aspirates from MM patients with relapsed refractory MM, CD73 inhibition by OP-5558 triggered activation of MM pDCs, evidenced by increased expression of CD40/CD83/CD86 (1.2-1.5-fold, OP-5558-treated versus untreated; p < 0.05; n=3). This inhibition of CD73 reversed immunosuppression in MM BM. Specifically, CD73 inhibitor OP-5558 stimulated T-cell activation, associated with increased CD69 cell surface expression on CD3 + T-cells (CD69 MFI:20% increase, treated versus control; p = 0.0031; n = 3). Moreover, CD8 + T-cells from these co-cultures enhanced cytolytic activity against patient MM cells, significantly decreasing autologous MM cell viability (mean 42% decrease in viability; treated versus control; p=0.014; n=5). Of note, OP-5558 treatment did not directly affect viability of MM cells when treated in isolation, indicating that the observed decreased viability occurs via enhanced cytotoxic T-cell activity. Importantly, we show that OP-5558 triggered significant MM cell lysis even within autologous MM bone marrow mononuclear cell (BMNC) cultures, confirming that CD73 inhibition restores MM-specific cytolytic activity of autologous patient T-cells in the MM BM microenvironment. (mean 37% decrease in viability; treated versus control; p=0.009; n=3). Conclusions: This study therefore demonstrates that: 1. CD73-mediated adenosine activity suppresses the cytolytic activity of T-cells against tumor cells in the MM BM milieu; and 2. CD73 inhibition can overcome immune suppression and restore lysis of MM cells by autologous T-cells. A clinical trial of potent, selective, orally bioavailable CD73 inhibitor ORIC-533 will examine the utility of CD73 inhibition to improve outcome in patients with relapsed refractory MM. Disclosures Junttila: ORIC Pharmaceuticals: Current Employment. Sutimantanapi: ORIC Pharmaceuticals: Current Employment. Chen: ORIC Pharmaceuticals: Current Employment. Warne: ORIC Pharmaceuticals: Current Employment. Chang: ORIC Pharmaceuticals: Current Employment. Blank: ORIC Pharmaceuticals: Current Employment. Wu: ORIC Pharmaceuticals: Current Employment. Moore: ORIC Pharmaceuticals: Current Employment. Ndubaku: ORIC Pharmaceuticals: Current Employment. Zavorotinskaya: ORIC Pharmaceuticals: Current Employment. Nadeem: GSK: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Friedman: ORIC Pharmaceuticals: Current Employment. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

Androgen deprivation therapy (ADT) with or without antiandrogens such as enzalutamide is the mainstay of treatment in advanced prostate cancer (PC). However, new therapeutic strategies are needed for patients who relapse on antiandrogen therapy. The glucocorticoid receptor (GR) has been implicated as a potential antiandrogen bypass mechanism. We previously showed that ORIC-101, a potent and selective, orally bioavailable small molecule GR antagonist, reverses GR-mediated resistance to enzalutamide in preclinical PC models. In the clinic, we are evaluating ORIC-101 in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Activation of the PI3K/AKT pathway through PTEN loss is a poor prognostic factor and another potential resistance mechanism to anti-hormone directed therapies. Reciprocal crosstalk between androgen signaling and PI3K/AKT pathway activation forms the basis for the clinical development of AKT inhibitors (AKTi) in combination with ADT or antiandrogens (NCT03072238, NCT04087174). In this study, we tested preclinically whether activated GR confers resistance to the combination of AKT inhibitors with enzalutamide and whether co-treatment with ORIC-101 reverses GR-mediated resistance to an enzalutamide/AKTi doublet. We first evaluated the effects of enzalutamide plus AKTi treatment on GR levels, GR target genes and activity in three prostate cancer cell lines: PTEN-null line LNCaP and PTEN-wildtype lines VCaP and CWR22PC. We tested three AKT inhibitors: the ATP-competitive inhibitors ipatasertib (GDC-0068) and capivasertib (AZD5363), in addition to the allosteric inhibitor MK2206, in CSS media and 10% FBS, with or without supplementation of the synthetic glucocorticoid, dexamethasone. An increase in the protein and mRNA levels of GR and GR target genes was observed after 7 days of enzalutamide treatment. In the AKTi plus enzalutamide setting, AKT inhibition did not block the enzalutamide-mediated GR upregulation in either PTEN-null or PTEN-wildtype cells. We next evaluated whether increased GR activity mediates resistance to the enzalutamide/AKTi doublet with ipatasertib or capivasertib, and whether this resistance can be reversed with ORIC-101. GR activation with dexamethasone promoted tumor cell growth and androgen-regulated gene expression in the presence of enzalutamide and ipatasertib or capivasertib, using a 21-day proliferation assay in CWR22PC cells. ORIC-101 was able to reverse these dexamethasone-induced effects, confirmed by reduced secreted PSA levels and cell number measured at end of study. These findings indicate that GR upregulation and activation, an established resistance mechanism for antiandrogens, may drive resistance when combined with an AKT inhibitor, and the GR antagonist ORIC-101 is able to overcome this resistance and restore antitumor activity. Citation Format: Shravani Barkund, Haiying Zhou, Lori S. Friedman, Anneleen Daemen. GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1420.

Adenosine is recognized for its anti-inflammatory and immunosuppressive properties. Signaling through adenosine receptors expressed on immune cells initiates a cascade that diminishes activation and proliferation of cytotoxic T-cells, impairs activity of natural killer cells and CD4+ effector T-cells, and promotes the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, converts AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many solid tumors and may correlate with unfavorable clinical outcomes; therefore, reducing the level of adenosine via inhibition of CD73 is a strategy for enhancing antitumor immune responses and overcoming therapeutic resistance. We have developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73. ORIC-533 is highly selective for CD73 and exhibits picomolar potency in biochemical and cellular assays, blocking adenosine production from AMP. Expression of CD73 on immune cells is sufficient to drive immunosuppression in vitro when AMP is present. We have previously shown that ORIC-533 can rescue activation of CD8+ T- cells exposed to AMP and restore proliferation, cytokine secretion and CD25 expression at low nanomolar concentrations. To understand the role of CD73 in dendritic cell (DC) priming of helper CD4+ T-cells, we utilized co-culture of allogeneic immature DC and CD4+ T-cells, where AMP to adenosine conversion severely suppresses activation of helper CD4+ T-cells. Treatment with ORIC-533 completely restored activation of CD4+ T-cells in this setting. To better define the biologically relevant AMP concentrations likely to be encountered in tumors, we determined the concentration of AMP in a panel of primary patient tumors. We discovered that intratumoral AMP levels can reach up to 500 micromoles, emphasizing the requirement for CD73 inhibitors to be efficacious in a high AMP environment. We previously demonstrated that nanomolar concentrations of ORIC-533, unlike other CD73 inhibitors tested, can efficiently rescue cytotoxic T-cell function in the presence of millimolar AMP concentrations. Surprisingly, in these studies we found that inhibitors of adenosine receptors, A2A or A2A/B, can only rescue CD8+ T-cell function in the context of low micromolar AMP or adenosine and thus are likely to be ineffective in patient tumors with high AMP. Taken together, these preclinical results indicate the CD73 inhibitor ORIC-533 has best-in-class properties in reversing immunosuppression in tumors. ORIC-533 is currently undergoing GLP studies to support an IND filing in the first half of 2021. Citation Format: Dena Sutimantanapi, Chelsea Chen, Bob Warne, Alex Pankov, Jae Chang, Lori Friedman, Melissa Junttila, Tatiana Zavorotinskaya. Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB163.

The polycomb repressive complex 2 (PRC2) is responsible for the methylation of histone 3 at lysine 27 (H3K27) which leads to long-term transcriptional silencing. Through this epigenetic chromatin silencing mechanism, PRC2 plays a key role in regulating cellular functions such as cell growth and differentiation. PRC2 comprises three core subunits: the catalytic subunit enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12). EED directly interacts with histone H3K27me3 and is essential for the histone methyltransferase activity of PRC2. PRC2 dysregulation occurs in multiple solid tumors and hematological malignancies, resulting in elevated levels of PRC2 activity and H3K27 trimethylation, and has been linked to poor prognosis in patients with metastatic prostate cancer. First-generation PRC2 inhibitors which target EZH2 have demonstrated clinical activity in several cancers, yet the pharmacological and ADME properties of these compounds require high doses that only achieve partial target inhibition at clinically active levels and exhibit drug-drug interaction (DDI) liabilities. As an alternative strategy to fully inhibit the PRC2 complex, we developed ORIC-944, a potent and highly selective allosteric inhibitor of PRC2 via binding the EED subunit. This unique EED targeting strategy can more completely inhibit PRC2, for example, in the presence of innate or acquired resistance mutations in EZH2, and by addressing the potential compensatory escape mechanism of EZH1-driven tumor growth. ORIC-944 has potential best-in-class drug properties compared to first generation PRC2 inhibitors, including superior potency and improved DDI liabilities. In diffuse large B-cell lymphoma (DLBCL) xenografts in vivo, ORIC-944 significantly depleted H3K27 trimethylation and induced tumor regressions in a dose-dependent manner. ORIC-944 demonstrated strong tumor growth inhibition as a single agent with once daily oral dosing in both enzalutamide-responsive and enzalutamide-resistant prostate cancer models. ORIC-944 caused a significant reduction in tumor cell proliferation and anti-apoptotic signaling, as measured by Ki67 and survivin, respectively. Moreover, in PK/PD assessments, ORIC-944 strongly depleted H3K27me3 in treated tumors. These in vivo studies established that effective single agent inhibition of PRC2 via EED results in decreased tumor cell growth in PRC2-dependent prostate cancer models. In summary, ORIC-944 is a potent, highly selective, allosteric, orally bioavailable PRC2 inhibitor via the EED subunit that represents a differentiated strategy to block PRC2 activity in selected cancers. We are developing ORIC-944 as a best-in-class PRC2 inhibitor for the treatment of patients with advanced prostate cancer and expect to file an IND in the second half of 2021. Citation Format: Anneleen Daemen, Jessica D. Sun, Aleksandr Pankov, Frank L. Duong, Natalie Yuen, Shravani Barkund, Shelly Kaushik, Jae H. Chang, David M. Briere, Niranjan Sudhakar, Andrew Calinisan, Aaron Burns, John M. Ketcham, Matthew A. Marx, Peter Olson, James G. Christensen, Melissa R. Junttila, Lori S. Friedman. ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1131.

Genomic insertions within exon20 of both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are oncogenic drivers most commonly found in non-small cell lung cancer (NSCLC) but also occurring in multiple other tumor types. Exon20 insertions render the receptors resistant to currently approved inhibitors, giving patients with tumors harboring such insertions a worse prognosis than with other activating EGFR mutations. Moreover, approximately one-third of patients with exon20 insertion mutations may develop central nervous system (CNS) metastases over the course of their disease. To address this unmet medical need, ORIC-114, a brain penetrant, orally bioavailable, irreversible small molecule inhibitor was designed to target exon20 insertions in EGFR and HER2. ORIC-114 is highly selective to the EGFR family of receptors, showing superior kinome selectivity compared with other reported exon20 inhibitors. In biochemical assays ORIC-114 displays low nanomolar potency, and importantly has enhanced potency on the EGFR exon20 NPG insertion relative to wildtype EGFR protein. ORIC-114 also demonstrates low nanomolar potency across exon20 insertion variants using cell-based assays measuring EGFR phosphorylation, downstream signaling and cell viability. ORIC-114 is readily brain available across multiple preclinical species hence studies were undertaken to investigate activity in both subcutaneous and intracranial tumor models. Herein, we explored the in vivo activity of ORIC-114. Consistent with our in vitro findings, robust activity was observed in EGFR exon20 patient-derived xenograft models using once daily oral administration, with greater than 90% tumor growth inhibition in the absence of body weight loss. Moreover, these significant antitumor effects correlate with decreased pharmacodynamic response as measured by phosphorylated EGFR in terminal tumors. To investigate whether the brain-penetrant attributes of ORIC-114 translate into therapeutic CNS activity, we utilized an intracranial luciferase-labeled EGFR mutant cell line model. Once daily oral administration of ORIC-114 significantly regressed established intracranial NSCLC tumors, demonstrating greater efficacy than TAK-788, commensurate with the superior brain to plasma exposure of ORIC-114. Taken together, these data establish ORIC-114 as a selective, irreversible, and brain penetrant EGFR inhibitor, making it a promising therapeutic candidate for development in patients with tumors harboring EGFR and HER2 exon20 insertions, including those with CNS metastases. ORIC-114 is anticipated to enter a global Phase 1/2 tumor-agnostic trial in genetically defined cancers in the second half of 2021. Citation Format: Melissa R. Junttila, Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Jihye Yoo, Youngyi Lee, Dong-Hyuk Seo, Jung Beom Son, Daekwon Kim, Hwan Geun Choi, Nam Doo Kim, Akash Das, Dena Sutimantanapi, Tatiana Zavorotinskaya, Chelsea Chen, Jae Chang, Matthew Panuwat, Lori Friedman. ORIC-114, a brain penetrant, orally bioavailable, irreversible inhibitor selectively targets EGFR and HER2 exon20 insertion mutants and regresses intracranial NSCLC xenograft tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1466.

The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome–CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.

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.

3110 Background: Preclinical studies have shown that activation of the glucocorticoid receptor (GR) leads to resistance to chemotherapeutics (eg taxanes) and antiandrogens across multiple tumor types, while GR inhibition enhances therapeutic efficacy. ORIC-101 is a potent, selective, and orally bioavailable small molecule GR antagonist undergoing clinical development in combination with nab-paclitaxel in patients with advanced solid tumors and in combination with enzalutamide in patients with metastatic prostate cancer. Methods: 21 patients were enrolled in the dose escalation portion of the phase 1b study, which evaluated both intermittent (5 days on, 2 days off for 21 days) and continuous dosing regimens of ORIC-101 across 5 cohorts (NCT03928314). Tumor tissue was obtained pre-treatment for 19 out of 21 patients, and on study or at the end of treatment for 11 patients. GR protein status was retrospectively evaluated using a proprietary IHC assay optimized for staining nuclear GR in the epithelial compartment of major tumor type tissues. Biopsies were also profiled with RNA-seq to evaluate a proprietary GR activation signature as a potentially predictive and pharmacodynamic (PD) biomarker. Blood-derived peripheral blood mononuclear cells (PBMCs) were collected for 20 patients along with the pre-treatment biopsy, in the morning of days 1, 5 and/or 8 of Cycle 1, and 2.5 or 6 hours after ORIC-101 administration. Blood cortisol levels were also simultaneously measured. PD modulation in PBMCs was assessed by RT-qPCR for biomarkers FKBP5, GILZ and PER1, selected for their consistent stimulation by GR and reversal with ORIC-101 in preclinical studies and observed PD modulation in healthy volunteers administered ORIC-101. Results: Nuclear GR protein was detected in most pre-treatment biopsies regardless of tumor type, and on treatment reduction of GR protein was observed across dose levels. At physiological systemic cortisol levels, ORIC-101 demonstrated PD suppression in PBMCs on days 1, 5 and 8 in the majority of patients. Cortisol levels increased post-dose in these patients due to negative feedback between cortisol and GR. Steady-state target engagement was not consistently demonstrated with the intermittent regimen. In healthy volunteer studies of ORIC-101, steady-state target suppression was consistently achieved after 7 consecutive daily doses of 200 or 350 mg of ORIC-101. Thus, continuous ORIC-101 administration was selected as the recommended phase 2 dose (RP2D) regimen, aimed at achieving sustained GR suppression for optimal chemotherapy re-sensitization. Conclusions: Biomarker data from patients enrolled in the phase 1b study provide evidence of on-target tumor cell eradication and PD modulation and support the RP2D and the tumor types selected for the ongoing dose expansion portion of the study. Clinical trial information: NCT03928314.

PI3K plays a key role in cellular metabolism and cancer. Using a mass spectrometry–based metabolomics platform, we discovered that plasma concentrations of 26 metabolites, including amino acids, acylcarnitines, and phosphatidylcholines, were decreased in mice bearing PTEN-deficient tumors compared with non–tumor-bearing controls and in addition were increased following dosing with class I PI3K inhibitor pictilisib (GDC-0941). These candidate metabolomics biomarkers were evaluated in a phase I dose-escalation clinical trial of pictilisib. Time- and dose-dependent effects were observed in patients for 22 plasma metabolites. The changes exceeded baseline variability, resolved after drug washout, and were recapitulated on continuous dosing. Our study provides a link between modulation of the PI3K pathway and changes in the plasma metabolome and demonstrates that plasma metabolomics is a feasible and promising strategy for biomarker evaluation. Also, our findings provide additional support for an association between insulin resistance, branched-chain amino acids, and related metabolites following PI3K inhibition. Mol Cancer Ther; 15(6); 1412–24. ©2016 AACR.

Background: Treatment of BC is conventionally based on the presence/absence of ER/PR or HER2 status of the primary tumor. We have enriched this approach by including major genetic and proteomic changes in tumors of individual patients in order to develop a better treatment-rationale based on an alteration driven signaling algorithm. Methods: Genomic and proteomic data from 75 BC patients seen in our center were retrospectively analyzed. Patients were re-biopsied after consultation and samples were characterized (IHC for ER, PR, and HER2; FFPE samples for genomic [Foundation Medicine] and proteomic analyses [Theranostics]). In vivo studies were conducted using xenograft models. Results: Although alterations of PIK3CA, PIK3R1, AKT, PTEN, MDM2, MDM4, TSC1, mTOR and RICTOR are most frequently observed in our patients, there is a distinct pattern of alteration(s) of the PI3K pathway genes in different subtypes of BC. A total of 76 genes were altered in 48 ER+BC patients. In 79% of ER+BC patients the above mentioned PI3K pathway genes were altered. Analyzing the set of alterations of genes in individual patients, we observed that within these 48 patients 25% exhibited alterations in more than one node of the pathway; the most common combination (alterations) being the amplification/mutation of PIK3CA with the amplification of MDM2/4 genes. The percentage of patients belonging to HER2+ & TNBC exhibiting similar alterations in the PI3K pathway genes were significantly lower (∼40%). Our previous in vivo studies demonstrated that GDC-0980 and BEZ235 enhanced the antitumor activity of ABT888 plus carboplatin in TNBC or trastuzumab in HER2+ BC respectively and blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, pS6RP and CD31. Mechanistically the action of the PI3K-mTOR pathway targeted drug(s) was tested using cell line based models of BC subtypes pertaining to their respective genomic alterations. A combination of a pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) blocked proliferative signals and enhanced apoptosis (cleaved caspase3) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells respectively as demonstrated by WB, flow cytometry, cell proliferation, viability and cytotoxicity assays. A recent study demonstrated that exposure to chemotherapy induced a phenotypic shift or cell state transition towards a transient CD44Hi/CD24Hi chemotherapy-tolerant state, leading to the activation of downstream non-receptor tyrosine kinase signaling towards an emerging adaptive resistance (Goldman et al., Nature Comm. 2015). Hence drug combination(s) are being tested for their effect on CD44/CD24 expression levels, results of which will be presented in the meeting. Conclusion: Plotting the genetic alterations from the patient on the signaling landscape will be useful in cracking the code leading to improved treatment options. Patient specific in-depth plotting of genetic alterations of the PI3K-mTOR pathway and the relevance of these alterations in the context of (1) mechanisms of PI3K-mTOR pathway targeted drugs and (2) cell signaling are critical in determining choice of drugs in BC subtypes. Citation Format: Carlson JH, Krie A, Williams C, Sun Y, Lin X, Williams K, Klein J, Friedman L, De P, Dey N, Leyland-Jones B. Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-04.

Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kβ relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).

Letrozole is a commonly used treatment option for metastatic hormone receptor-positive (HR+) breast cancer, but many patients ultimately relapse. Due to the importance of phosphoinositide-3 kinase (PI3K) in breast cancer, PI3K inhibitors such as taselisib are attractive for combination with endocrine therapies such as letrozole. Taselisib was evaluated as a single agent and in combination with letrozole in a breast cancer cell line engineered to express aromatase. The combination of taselisib and letrozole decreased cellular viability and increased apoptosis relative to either single agent. Signaling cross-talk between the PI3K and ER pathways was associated with efficacy for the combination. In a secreted factor screen, multiple soluble factors, including members of the epidermal and fibroblast growth factor families, rendered breast cancer cells non-responsive to letrozole. It was discovered that many of these factors signal through the PI3K pathway and cells remained sensitive to taselisib in the presence of the soluble factors. We also found that letrozole resistant lines have elevated PI3K pathway signaling due to an increased level of p110α, but are still sensitive to taselisib. These data provide rationale for clinical evaluation of PI3K inhibitors to overcome resistance to endocrine therapies in ER+ breast cancer.

Introduction: Triple Negative (TN) BC has a limited benefit from conventional therapy and a few options for targeted therapy like PARP inhibitor. We reported that doubling down on the PI3K-mTOR pathway enhances the antitumor efficacy of PARP inhibitor in TNBC (Neoplasia, 2014). Tumor cells with distinctive stem cell (CSC) like properties exist in carcinomas including breast and CD44+CSC marker expression correlates with decreased survival (Fillmore and Kuperwasser, 2008). In TNBC the intratumoral presence of stem cell population is associated with increased aggressiveness. Among many CSC markers, CD44 is best known to have a prognostic role (Collina et al., 2015) and the PI3K/E2F1 pathway has been identified as a potential signaling link to HA/CD44 activation. Hypothesis: We hypothesized that xenografts of TNBC cell lines bearing stem cell property will respond in a characteristic way to GDC0980 in combination with ABT888 plus carboplatin. Methods:Athymic mice bearing BRCA-competent TNBC xenograft tumors were used to test the combination of GDC-0980 with ABT888 plus carboplatin. Mechanism-based in vitro studies were conducted to understand the mode of action of the drugs. IHC for CD44 and CD44v6 was standardized in positive tumor-controls and identified in (1) BC TMAs, (2) TNBC cell lines and (3) patients from our Avera cohort. Finally, the CD44-expression in tumors of different arms of the xenograft study (MDA-MB231 and MDA-MB468) was evaluated independently by a pathologist who was unaware of the study design. Results: CD44 was identified primarily in the membrane and to a lesser extent in the cytosol of tumor cells of BC patients (TMA and Avera cohort), TNBC cell lines and xenograft-tumors. GDC-0980 in combination with ABT888 plus carboplatin blocked the growth of established xenograft tumors by 80-90% with a concomitant decrease in Ki67 IHC-levels. Membrane expression of CD44 inversely correlated to tumor sizes which significantly reduced in response to drug combinations. Mechanistically, GDC-0980 treatment led to DNA damage (increased pgH2AX), gain in PAR and a subsequent sensitization of BRCA-competent TNBC cells to ABT888 plus carboplatin with a time-dependent (1) decrease in proliferation signals (pAKT), PAR/PARP ratios, PAR/pgH2AX ratios, live/dead cell ratios, and clonogenic 3D growth & (2) increase in apoptosis markers (cleaved-caspase3, cleaved-PARP and annexinV positivity). The relationship between expression of CD44 and CD44v6 in the xenograft tumors the following treatment is being worked out which will be presented at the meeting. Significance: Our findings demonstrated that PARP inhibitor plus carboplatin in combination with dual PI3K-mTOR inhibition effectively reduced the CD44+CSC population in TNBC xenograft tumors. In a BRCA-competent model, PI3K-pathway inhibition not only enhanced the antitumor activity of ABT888 plus carboplatin by inhibiting proliferation and tumor-induced angiogenesis but also by decreasing CD44+CSC. We present the first mechanism-based study to demonstrate the integral role of PI3K-mTOR pathway and DDR pathway in the control of CSC surface marker in orchestrating antitumor actions of PARP inhibitor in TNBC. Citation Format: Lin X, Sun Y, Carlson JH, Williams C, Krie A, Friedman L, De P, Dey N, Leyland-Jones B. Dynamics of cancer stem cell surface marker, CD44 and CD44v6 in the antitumor efficacy of PARP inhibitor in combination with PI3K pathway inhibitor in TNBC xenograft model [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 P6-08-03.

Activating mutations in PIK3CA are among the most significant oncogenic events across all cancers, making it an important target for drug development. Yet the application of PI3K inhibitors in the clinic has been limited by the difficulty of achieving an adequate therapeutic window, due to the critical role that PI3K signaling plays in normal physiologic processes, such as glucose homeostasis. In theory, the therapeutic window could be improved if it were possible to design mutant selective inhibitors, as has been demonstrated with other oncogenes such as EGFR. However, unlike EGFR, the most predominant PIK3CA activating mutations do not reside in the kinase active site, presenting a major challenge for rational structure-based design. Nevertheless, it was recently shown that the PI3K inhibitor taselisib is able to achieve modest levels of mutant selectivity both across cancer lines as well as in cell lines that were engineered to express mutant or wild-type PIK3CA. Taselisib was also shown to selectively induce degradation of mutant versus wild-type PIK3CA, leading to the speculation that this degradation may be responsible for the observed selectivity. In order to better understand the origins of mutant selectivity for taselisib and several other PIK3CA inhibitors, we assessed these inhibitors in a variety of biophysical and biochemical assays under conditions designed to mimic physiologic settings. In parallel, we also investigated the mechanistic basis of this selectivity in our engineered cell lines. Our results are consistent with the hypothesis that selective degradation of mutant PIK3CA is the predominant mechanism underlying mutant selectivity for this class of PIK3CA active site inhibitors. This abstract is also being presented as Poster B03. Citation Format: Lan Nguyen, Kyle Edgar, Kyung Song, Stephen Schmidt, Victorai Schutz, Noriko Ishisoko, Eric Torres, Akash Das, Divya Murali, Steve Sideris, Timothy Wendorff, Matt Saabye, Hans Purkey, Jawahar Sudhamsu, Steven Staben, Emily Hanan, Georgia Hatzivassiliou, Lori Friedman, Nicholas F. Endres. Selective degradation of mutant PIK3CA promotes increased mutant specificity in a subset of PI3K ATP-competitive inhibitors [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 PR03.

The 5' ecto-nucleotidase CD73 is dynamically expressed in distinct biological contexts as a means to increase the adenosine metabolite, which functions to limit immune activation and prevent excessive inflammation. CD73 activity is required for the final step of adenosine production. In the context of tumors, excessive CD73-mediated adenosine generation impairs anti-tumor immune responses. Here, we show that an orally bioavailable small molecule inhibitor of CD73 can prevent adenosine generation, relieve adenosine-driven immunosuppression within the tumor in vivo and generate anti-tumor efficacy. In vitro analysis confirmed the substantial suppressive effects of adenosine on murine CD8+ T cells, cross-presenting dendritic cells, and macrophages. NECA, a stable analog of adenosine, impaired immature bone-marrow precursor differentiation into CD103+ dendritic cells, a subset which is critical for tumor-derived antigen presentation to naïve T cells. Interestingly, NECA also inhibited the ability of macrophages to upregulate the M1 marker, CD80, in both the presence and absence of additional polarizing stimuli. Furthermore, OR-558, a CD73 small molecule inhibitor, could fully inhibit CD73-mediated adenosine production and completely restore T cell activation at sub-nanomolar concentrations. Prior work in CD73 germline knock-out animals has shown that EG7 syngeneic tumor growth is highly dependent on CD73. Building on this knowledge, we next we sought to evaluate CD73 inhibition on AMP to adenosine conversion ex vivo in EG7 syngeneic tumor extracts and indeed observed that OR-558 treatment could significantly impair adenosine production within the tumor milieu. This finding translated to the in vivo tumor setting, where single agent anti-tumor efficacy was observed with continuous OR-558 treatment. Anti-tumor efficacy coincided with significantly decreased intra-tumoral adenosine levels and immune modulation, such as increased intratumoral T cell activation, dendritic cell maturation, and M1 macrophage polarization. Lastly, we determined the bioavailability of orally dosed OR-558 in EG7 tumor-bearing mice and found that significant single agent anti-tumor efficacy was also achievable with once-daily oral dosing of OR-558. Taken together, these data demonstrate that adenosine has pleiotropic immunosuppressive effects and inhibition of CD73-mediated adenosine production in vivo is sufficient to reverse these immune effects and enhance anti-tumor immunity. These results support clinical development of orally bioavailable CD73 small molecule inhibitors. Citation Format: Todd Metzger, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Melissa Junttila, Hiro Kawai, Wayne Kong, Jared Moore, Johnny Pham, Yosup Rew, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Kejia Wu, Chien-Hung Yeh, Natalie Yuen, Tatiana Zavorotinskaya. An orally bioavailable inhibitor of CD73 reverts intratumoral immunosuppression and promotes anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-115.

CD73 has emerged as an attractive target for cancer immunotherapy. CD73 (ecto-5-nucleotidase) is a glycosyl phosphatidyl inositol (GPI)-anchored cell surface protein and catalyzes the hydrolysis of AMP into immunosuppressive adenosine and inorganic phosphate. CD73 is widely overexpressed in tumor microenvironments (TME) of many cancers, resulting in elevated levels of extra cellular adenosine (ADO). ADO plays a critical role in tumor progression through immune suppression, chemotherapy resistance, metastasis and angiogenesis. Therefore, reducing the level of adenosine via inhibition of CD73 has become a potential strategy for treating cancers. Here we report our medicinal chemistry efforts in developing orally available small molecule CD73 inhibitors. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (APCP) with CD73, we designed a novel series of monophosphate CD73 inhibitors, which are highly potent and orally bioavailable. In preclinical studies, OP-5244 inhibited ADO production completely in human cancer cells and CD8+ T cells. It also showed dose-dependent inhibitory effects on CD73 activity in various tumors ex vivo. Furthermore, OP-5244 showed PK/PD efficacy through lowering of ADO/AMP ratio and reversal of immunosuppression in vivo. Citation Format: Xiaohui Du, Brian Blank, Brenda Chan, Xi Chen, Yuping Chen, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd Metzger, Jared Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen, Tatiana Zavorotinskaya. Orally available small molecule CD73 inhibitor reverses immunosuppression through blocking of adenosine production [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1037.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and one of the most lethal cancers, with a 5-year survival rate of 8%. Chemotherapy remains the main treatment option for patients with advanced and metastatic tumors. However, intrinsic resistance to chemotherapeutics and lack of effective targeted therapies are major factors contributing to the dismal prognosis. Activation of glucocorticoid receptor (GR) signaling confers resistance to chemotherapy in solid tumors, making GR antagonism an attractive combination treatment strategy to overcome chemotherapeutic resistance. ORIC-101 is a novel selective GR antagonist under clinical evaluation in combination with anticancer therapies. We previously reported that ORIC-101 can sensitize triple-negative breast cancer cells to chemotherapy by reversing multiple GR-modulated pathways. Here, we investigate the effects of ORIC-101 in preclinical models of PDAC. Employing in vitro functional assays and gene expression analysis, ORIC-101 exhibited chemo-potentiation in a panel of PDAC cell lines. Biomarker analysis of transcription activity following ORIC-101 treatment in vitro confirmed pharmacodynamic (PD) modulation of core GR target genes, indicative of suppressed GR signaling. Extending these findings in vivo across several PDAC xenograft models (SW1990, BxPC3, and HPAC) revealed that ORIC-101 blocks GR transcriptional activity induced by dexamethasone in a dose-dependent manner. The PD modulation observed in vivo correlated with ORIC-101 plasma exposure. Consistent with ORIC-101's ability to suppress GR activity, in vivo efficacy studies in combination with paclitaxel significantly inhibited PDAC tumor growth compared to the paclitaxel control group. These results in PDAC preclinical models support the clinical development of ORIC-101 in overcoming GR-driven chemoresistance. A phase 1b study of ORIC-101 in combination with nab-paclitaxel in patients with advanced or metastatic solid tumors is ongoing (NCT03928314). Citation Format: Jessica D. Sun, Haiying Zhou, Wayne Kong, Natalie Yuen, Frank L. Duong, Shravani Barkund, Aleksandr Pankov, Dan McWeeney, Qiuping Ye, Omar Kabbarah, Lori S. Friedman, Anneleen Daemen, Melissa Junttila. ORIC-101 overcomes glucocorticoid receptor-mediated chemoresistance in pancreatic cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4123.

Adenosine has emerged as a key immunosuppressive metabolite within the tumor microenvironment (TME). A persistently elevated concentration of adenosine in TME can impair immune control of tumor growth by diminishing cytotoxic T-cell responses and function of natural killer cells while augmenting the suppressive activity of myeloid and regulatory T-cells. The ectonucleotidase, CD73 mediates the final dephosphorylation step in the conversion of extracellular ATP to adenosine. Overexpression of CD73 is observed in many solid tumors and correlates with unfavorable clinical outcome. We aimed to overcome adenosine-driven immunosuppression by developing an orally bioavailable, best-in-class, small molecule inhibitor of CD73. Here, we show superior potency of our selective AMP-competitive CD73 inhibitor in blocking adenosine generation by multiple cell types. CD73 inhibitor activity was assessed by directly measuring the generation of adenosine from AMP by LC-MS/MS. In CD73 expressing cells, potent and complete inhibition of CD73 ectonucleotidase activity was observed. To test the functional consequence of CD73 inhibition, we interrogated the effects of CD73 inhibitors on CD8+ T-cell function upon exposure to AMP. CD73 expressed on T-cells is sufficient to drive adenosine generation from AMP resulting in severely suppressed proliferation, diminished production of inflammatory cytokines and reduced expression of activation markers. Our inhibitors successfully counteracted the effects of AMP and completely rescued all aspects of CD8+ T-cell activation. Interestingly, we found that the concentration of AMP in tumors may vary from micromolar to millimolar levels, underscoring the necessity of AMP-competitive CD73 inhibitors to be efficacious in a high AMP environment. We demonstrated that nanomolar concentrations of our CD73 inhibitors can efficiently rescue T-cell function in the presence of millimolar AMP concentrations. We are currently conducting biophysical studies to better understand this unique property of our inhibitors. We next aimed to identify an adenosine responsive gene signature in cytotoxic T-cells. RNA sequencing analysis of primary anti-CD3/28/2 activated CD8+ T-cells from human peripheral blood mononuclear cells revealed a unique set of genes persistently and similarly altered in response to both adenosine and AMP. Finally, we demonstrated that CD73 inhibitors can completely block gene expression changes of this AMP/adenosine-response signature. Discovery of an AMP/adenosine-response gene signature may help identify patients whose tumors harbor elevated adenosine signaling and would benefit from an orally bioavailable small molecule inhibitor of CD73 to reverse immunosuppression. Citation Format: Tatiana Zavorotinskaya, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd C. Metzger, Jared T. Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen. CD73 inhibition with a novel orally bioavailable small molecule blocks adenosine production and rescues T-cells activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1023.

Preclinical studies have shown that GR activation leads to decreased response to antimetabolites, taxanes, and platinum agents, while GR inhibition enhances therapeutic efficacy. The novel, selective GR antagonist ORIC-101 is under clinical evaluation in combination with anticancer therapies. In this study we set out to assess which cancer types and chemotherapeutics are responsive to combination with ORIC-101. Specifically, we employed caspase assays, in vitro and in vivo tumor growth inhibition studies, and transcriptional profiling in a panel of preclinical models spanning triple-negative breast, ovarian, non-small cell lung cancers, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, sarcoma, and renal cell carcinoma. We found that ORIC-101 reverses GR-mediated antiapoptosis effects towards diverse chemotherapeutics, including paclitaxel, gemcitabine, and cisplatin, across the seven cancer types. Furthermore, ORIC-101 completely overcomes GR-driven chemoprotection and tumor growth in colony formation assays in vitro and in xenograft studies in vivo. At the molecular level, transcriptional profiling and pathway enrichment analysis showed that ORIC-101 fully reverses GR-activated pathways that are directly involved in drug resistance, such as epithelial-to-mesenchymal transition and antiapoptosis, supporting a pan-cancer role of GR as a mediator of therapy resistance. Altogether, ORIC-101, a selective and potent GR antagonist, overcomes resistance to common chemotherapeutics across multiple cancer models. Clinical evaluation of ORIC-101 in combination with nab-paclitaxel is currently ongoing in advanced solid tumors. Citation Format: Haiying Zhou, Shravani Barkund, Aleksandr Pankov, Ganapati Hegde, Wayne Kong, Padmini Narayanan, Jessica D. Sun, Omar Kabbarah, Lori S. Friedman, Anneleen Daemen. ORIC-101 overcomes resistance to diverse chemotherapeutics across cancer types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4121.