Potter, David A

Abstract Purpose: The neutralizing peptibody trebananib prevents angiopoietin-1 and angiopoietin-2 from binding with Tie2 receptors, inhibiting angiogenesis and proliferation. Trebananib was combined with paclitaxel±trastuzumab in the I-SPY2 breast cancer trial. Patients and Methods: I-SPY2, a phase II neoadjuvant trial, adaptively randomizes patients with high-risk, early-stage breast cancer to one of several experimental therapies or control based on receptor subtypes as defined by hormone receptor (HR) and HER2 status and MammaPrint risk (MP1, MP2). The primary endpoint is pathologic complete response (pCR). A therapy “graduates” if/when it achieves 85% Bayesian probability of success in a phase III trial within a given subtype. Patients received weekly paclitaxel (plus trastuzumab if HER2-positive) without (control) or with weekly intravenous trebananib, followed by doxorubicin/cyclophosphamide and surgery. Pathway-specific biomarkers were assessed for response prediction. Results: There were 134 participants randomized to trebananib and 133 to control. Although trebananib did not graduate in any signature [phase III probabilities: Hazard ratio (HR)-negative (78%), HR-negative/HER2-positive (74%), HR-negative/HER2-negative (77%), and MP2 (79%)], it demonstrated high probability of superior pCR rates over control (92%–99%) among these subtypes. Trebananib improved 3-year event-free survival (HR 0.67), with no significant increase in adverse events. Activation levels of the Tie2 receptor and downstream signaling partners predicted trebananib response in HER2-positive disease; high expression of a CD8 T-cell gene signature predicted response in HR-negative/HER2-negative disease. Conclusions: The angiopoietin (Ang)/Tie2 axis inhibitor trebananib combined with standard neoadjuvant therapy increased estimated pCR rates across HR-negative and MP2 subtypes, with probabilities of superiority >90%. Further study of Ang/Tie2 receptor axis inhibitors in validated, biomarker-predicted sensitive subtypes is warranted.

Abstract Introduction: Immune checkpoint blockade (ICB) has clinical activity in triple negative breast cancer (TNBC) but is less effective in the ER+HER2- signature, where there is a cold immune microenvironment (IM) and regulatory T cells (Tregs) may suppress effector T cells. Agents that activate the IM by turning cold tumors hot may support ICB. The biguanides hexyl-benzyl-biguanide (HBB) and its bioisostere hexyl-(cuban-1-yl-methyl)-biguanide (HCB) are candidate agents to activate the IM because they potently inhibit biosynthesis of immunosuppressive epoxyeicosatrienoic acids (EETs) and EET-driven oxidative phosphorylation (OXPHOS), while blocking N-glycosylation of immune checkpoint (IC) proteins. We hypothesized that reversal of hypoxia by biguanides in the ovarian dependent ER+HER2- STAT1 KO SSM2ucd mouse mammary carcinoma (MC) model would suppress Tregs and promote effector T cells in the tumor IM. While the SSM2ucd model did not express immune checkpoint protein PD-L1 (B7-H1), it did express related IC proteins B7-H3 and B7-H4. We hypothesized that by inhibiting OXPHOS and reducing N-glycosylation of immune checkpoint proteins, HBB and HCB may promote efficacy of ICB. We chose the SSM2ucd model to test impact of HCB on the ER+ MC IM. Results: SSM2ucd cells exhibited longer tumor latency (60 days) than the basal 4T1 (10 days) and 67NR (20 days) mouse MC models. SSM2ucd tumor reimplantation shortened latency by more than half, to 20 days. Immunohistochemistry showed that B7-H3 and B7-H4 protein levels were 1.2 (P=0.001) and 1.3-fold (P=0.04) higher in reimplanted tumors vs. control. In SSM2ucd cells, HCB inhibited N-glycosylation of B7-H3 (P=0.01) by 35% and B7-H4 (P=0.02) by 45% and suppressed TGFβ induction of B7-H3 by 21% (P=0.02) and B7-H4 by 79% (P=0.001) at 24 hours, while 14,15-EET promoted N-glycosylation of B7-H3 (1.2-fold; P=0.03) and B7-H4 (1.3-fold; P=0.04) at 4 hours. Effects of HBB and HCB on anti-CD3 and anti-CD28 stimulated mouse splenocytes were assayed. The proliferative effects of HBB on CD4+ and CD8+ cells peaked at 12 uM (p<0.001) and for HCB at 6.25 uM (p<0.001). Tregs decreased with HCB at a threshold of 12 uM (p <0.001) and with HBB at 25 uM (P < 0.001) while 14,15-EET increased Tregs (1.2 fold; P=0.02) and suppressed the CD8+:Treg ratio (0.79; P=0.02). In the SSM2ucd mammary allograft model, although HCB 12mg/kg daily did not inhibit tumor growth, it reduced intratumoral hypoxia by 20% (P=0.01), increased CD8+ TIL by 4.4-fold (P=0.04), decreased Treg:CD4+ TIL ratio by 76% (P=0.02), and decreased Treg:CD8+ TIL ratio by 88% (P=0.01) relative to control. Conclusion: B7-H3 and B7-H4 expression inversely correlated with latency of ER+ MC and may represent targets for immune checkpoint antibodies and their drug conjugates. HCB, an inhibitor of OXPHOS and EET biosynthesis, reduced intratumoral hypoxia, increased CD8+ TIL and reduced the Treg:CD8+ ratio, potentially supporting ICB therapy of ER+ MC by turning cold tumors hot. Supported by CDMRP BCRP Grant BC180596, Award Number W81XWH-19-1-0099 Citation Format: Zhijun Guo, Jianxun Lei, Hrishi Venkatesh, David Owen, Adam Bass, Christine Cannon, Joshua McCarra, Brenda Koniar, Craig Flory, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John Hawse, Emmanuel S. Antonarakis, Emanuel F. Petricoin, Julia Wulfkuhle, Robert D. Cardiff, Elizabeth A. Ambrose, Gunda I. Georg, Kaylee L. Schwertfeger, Michael A. Farrar, Brad St. Croix, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB102.

Abstract Background: I-SPY 2 is a multicenter, phase 2 trial using response-adaptive randomization within molecular subtypes defined by receptor status and MammaPrint (MP) risk to evaluate novel agents as neoadjuvant therapy for women with high-risk breast cancer. Tucatinib is a potent HER2 (ErbB2) tyrosine kinase inhibitor, selective for HER2 vs. epidermal growth factor receptor (EGFR) and is active vs. brain metastases. Safety and efficacy of tucatinib combined with paclitaxel, pertuzumab, and trastuzumab are unknown and were tested in a planned 10 patient (pt) safety run-in of the I-SPY 2 trial. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only pts with tumors that were HER2+ by FISH were eligible for this treatment. Treatment included tucatinib (max dose 300 mg) BID for 12 weeks with weekly paclitaxel 80 mg/m2 and trastuzumab (2 mg/kg weekly following loading), and pertuzumab (420 mg every 3 weeks following loading), followed by doxorubicin/cyclophosphamide (AC) every 2 weeks x 4. The control arm was weekly paclitaxel and trastuzumab with pertuzumab for 12 weeks followed by AC every 2 weeks x 4. All pts undergo serial MR imaging and response at 3 & 12 weeks is combined with real time pCR data to estimate, and continuously update, the predicted pCR rate for each trial arm. The goal of the trial is to identify/graduate regimens with ≥85.% Bayesian predictive probability of success (i.e. demonstrating superiority to control) in a future 300-patient phase 3 neoadjuvant trial with a pCR endpoint. This run-in arm was conducted to determine safety of combining tucatinib with paclitaxel/trastuzumab/pertuzumab, monitoring special adverse events of interest including LFT elevations and gastrointestinal toxicities.Methods: The I-SPY 2 methods have been previously published. Results: 20 pts were evaluable in tucatinib treatment arm. The control arm included 329 historical controls enrolled since April 2010. The initial tucatinib dose was 300 mg BID. After enrollment of the first 8 pts, there were 3 pts with grade 3 LFT elevations, 2 pts with grade 2/3 diarrhea, 1 pt with grade 2 neutropenia, and 1 pt with grade 3 nausea. After this safety review, the tucatinib dose was lowered to 250 mg BID. Among 5 additional pts enrolled, 3 developed grade 2/3 LFT abnormalities. The protocol was then modified to tucatinib 150 mg BID days 1-28 and then 250 mg BID days 29-84; 7 pts were treated. Safety data were reviewed after 20 pts were enrolled; the arm was then suspended due to similar LFT elevations regardless of tucatinib dose reduction or schedule. 7 of 20 pts (35%) had reversible Grade 3 or higher ALT/AST elevation (Table). No pt met criteria for Hy’s Law. In terms of efficacy, 12 of 14 evaluable pts had > 80% reduction of tumor volume by 12 weeks, measured by MRI assessment of functional tumor volume (FTV). Conclusion: The goal of the run-in arm was to determine the safety of adding tucatinib to the combination of paclitaxel/trastuzumab/pertuzumab. The addition of tucatinib resulted in unacceptable but reversible LFT elevations despite tucatinib dose reduction. Tucatinib containing therapy resulted in >80% decline in tumor volume at 12 weeks in 86% of pts. Tucatinib showed a high level of activity when combined with paclitaxel/trastuzumab/pertuzumab, but the combination is not feasible. Table: Number of pts with grade 2, 3, and 4 LFT elevations by treatment schedule (highest grade per patient per event, ALT or Treatment scheduleGrade 2 LFT elevationGrade 3 LFT elevationGrade 4 LFT elevationTucatinib 300 mg BID030Tucatinib 250 mg BID210Tucatinib 150 mg BID days 1-28 followed by 250 mg BID days 29 to 84112 Citation Format: David A Potter, Erin Roesch, Christina Yau, Ruixiao Lu, Denise Wolf, Susan Samson, Debra Stafford, Kathy S Albain, Claudine Isaacs, Meghana Trivedi, Douglas Yee, Judy Boughey, Alexandra Thomas, A. Jo Chien, Nola Hylton, Wen Li, Angela DeMichele, Jane Perlmutter, W. Fraser Symmans, Dawn L Hershman, Michelle Melisko, Laura J van 't Veer, Amy Wilson, Smita M Asare, Donald A Berry, Richard Schwab, Hope S Rugo, Laura J Esserman. Evaluation of Tucatinib + (Paclitaxel + Pertuzumab + Trastuzumab) followed by AC in high-risk HER2 positive (HER2+) stage II/III breast cancer: Results from the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD8-07.

Abstract Purpose: This open-label, multicenter, phase IB/II study evaluated sapanisertib, a dual inhibitor of mTOR kinase complexes 1/2, plus exemestane or fulvestrant in postmenopausal women with hormone receptor–positive (HR+)/HER2-negative (HER2−) advanced/metastatic breast cancer. Patients and Methods: Eligible patients had previously progressed on everolimus with exemestane/fulvestrant and received ≤3 (phase IB) or ≤1 (phase II) prior chemotherapy regimens. Patients received sapanisertib 3 to 5 mg every day (phase IB), or 4 mg every day (phase II) with exemestane 25 mg every day or fulvestrant 500 mg monthly in 28-day cycles. Phase II enrolled parallel cohorts based on prior response to everolimus. The primary objective of phase II was to evaluate antitumor activity by clinical benefit rate at 16 weeks (CBR-16). Results: Overall, 118 patients enrolled in phase IB (n = 24) and II (n = 94). Five patients in phase IB experienced dose-limiting toxicities, at sapanisertib doses of 5 mg every day (n = 4) and 4 mg every day (n = 1); sapanisertib 4 mg every day was the MTD in combination with exemestane or fulvestrant. In phase II, in everolimus-sensitive versus everolimus-resistant cohorts, CBR-16 was 45% versus 23%, and overall response rate was 8% versus 2%, respectively. The most common adverse events were nausea (52%), fatigue (47%), diarrhea (37%), and hyperglycemia (33%); rash occurred in 17% of patients. Molecular analysis suggested positive association between AKT1 mutation status and best treatment response (complete + partial response; P = 0.0262). Conclusions: Sapanisertib plus exemestane or fulvestrant was well tolerated and exhibited clinical benefit in postmenopausal women with pretreated everolimus-sensitive or everolimus-resistant breast cancer.

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Δ) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Δ cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Δ cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Δ cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, β-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Δ cells with a decrease in G1 and increase in S and G2 fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Δ cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor. [Mol Cancer Ther 2008;7(11):3586–97]