Petricoin, Emanuel F.

Of the thousands of genes and substrates identified in KRAS-mutant signaling networks in pancreatic ductal adenocarcinoma (PDAC), more than 200 are transcription factors, implying extensive and complex transcriptional regulation. However, we observed that genetic suppression of the transcription factor MYC alone was sufficient to phenocopy the effect of KRAS suppression in signaling, growth, and metabolic processes in PDAC cells. We determined the gene transcription changes caused by acute suppression of MYC function in KRAS -mutant PDAC cell lines and performed dependency map and pathway analyses on the affected gene sets. The expression of 1685 genes was increased upon suppression of MYC, and this gene set may comprise the bulk of the MYC-regulated genes essential for PDAC growth. In contrast, the 1325 genes whose expression was inhibited may comprise a compensatory response to oncogenic stress, mediated in part by the GTPase RHO. MYC-dependent transcriptional activity was largely ERK dependent, and almost one-third of ERK-regulated genes were also regulated by MYC in PDAC cells. Furthermore, chemical proteomic profiling revealed MYC-regulated protein kinases that can be targeted therapeutically. Together, these data provide a molecular portrait of MYC-dependent signaling that encompasses potentially exploitable mechanisms for treating PDAC.

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: The efficacy of immunotherapy, including therapeutic strategies capable of modulating innate/adaptive immune resistance, varies greatly across tumor types. As the number of available immunotherapies accelerates, the study of predictive markers by IHC (e.g. PDL1 expression) is under intense investigation. However, staining protocol inconsistency, variation across scoring systems, and subjective interpretation of the immunostaining have produced conflicting results thus far. This work explored the role of Laser Capture Microdissection (LCM) coupled with Reverse Phase Protein Microarray (RPPA) as an alternative high throughput, quantitative, operator independent platform for measuring PDL1 expression across tumor types. Material and Methods: Pure epithelial cells were isolated via LCM from 178 samples including: 72 ovarian cancers (OC), 57 lung adenocarcinomas (LC), 30 metastatic breast cancers (MBC), and 19 pancreatic cancers (PC). PDL1 expression was measured on a continuous scale using quantitative RPPA based analysis. Each tumor type was processed and arrayed independently. Experimental samples and reference standards used for inter-assay normalization were printed in triplicates. Results: PDL1 expression varied greatly across tumor types. LC were characterized by the greatest intra-tumor fold dynamic range (> 35-fold), followed by OC (< 13-fold), MBC (< 4-fold), and PC (< 2-fold). PDL1 expression of 46/57 (80.7%) LC, 17/30 (56.7%) MBC, 6/19 (31.5%) PC, and 20/72 (27.8%) OC was greater than the population median of all tumors combined. Within the LC samples with PDL1 expression equal to the top quartile of the population, 10 (71.4%) were KRAS mutant lesions and 4 (28.6%) were WT tumors. Finally, amongst LC and PC harboring a KRAS mutation, PC showed an overall lower expression of PDL1 with only 2/19 (10.5%) cases been above the population median and none within the top quartile of the population. Conclusions: The LCM-RPPA workflow has the unique ability to capture immune checkpoint expression on a continuous quantitative scale as well as capture its broad dynamic range. Because RPPA is unconstrained by antigen retrieval issues as well as subjectivity of IHC interpretation, this approach may generate more accurate cut-point of therapeutic response prediction. Overall the dynamic range of PDL1 was broader in LC compared to other solid tumors, and LC had a much higher proportion of patients with tumors expressing high levels of PDL1. These quantitative differences may explain therapeutic efficacy of PDL1 inhibition across tumor types. Such speculative hypothesis should be further validated in prospective clinical trials. Finally, these preliminary data suggest that organ specific microenvironments more than specific driving mutations (e.g. KRAS) may strongly influence PDL1 expression in malignant lesions. Citation Format: Elisa Baldelli, Valerie Calvert, K. Alex Hodge, Maria Isabella Sereni, Guido Gambara, Eric B. Haura, Lucio Crino', Bryant Dunetz, Sergio Pecorelli, David J. Perry, Stephen P. Anthony, Nicholas Robert, Donald W. Northfelt, Mohammad Jahanzeb, Emanuel F. Petricoin, Mariaelena Pierobon. Quantitative measurement of PDL1 expression across tumor types using laser capture microdissection and reverse phase protein microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5656. doi:10.1158/1538-7445.AM2017-5656