PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used offlabel for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (À13% and À14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P ¼ 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.

BACKGROUND Curcumin has proven to be a potent antitumor agent in both preclinical and clinical models of colorectal cancer (CRC). It has also been identified as a ligand of the transcription factor known as the aryl hydrocarbon receptor (AHR). Our lab has identified the AHR as a mechanism which contributes to both tumorigenesis in a mouse model of inflammatory CRC as well an apoptotic target in vitro. Curcumin’s role as an AHR ligand may modulate its effects to induce colon cancer cell death, and this role may be enhanced via structural modification of the curcumin backbone. We sought to determine if the two piperidone analogs of curcumin, RL66 and RL118, exhibit more robust antitumor actions than their parent compound in the context of colorectal cancer in vitro. Moreover, to ascertain the ability of curcumin, RL66, and RL118 to activate the aryl hydrocarbon receptor and evaluate if this activation has any effect on CRC cell death. MATERIALS AND METHODS DLD1, HCT116, LS513, and RKO colon cell lines were propagated in vitro. Natural curcumin was obtained commercially, while RL66 and RL118 were synthesized and characterized de novo. Multi-well fluorescent/luminescent signal detection was utilized to simultaneously ascertain cell viability, cell cytonecrosis, and relative amounts of apoptotic activity. AHR activity was measured with a dual luciferase reporter gene system. Stable expression of siRNA interference was established in the HCT116 cell lines to create AHR “knock down” cell lines. RESULTS Both RL66 and RL118 proved to be more potent antitumor agents than their parent compound curcumin in all cell lines tested. The majority of this cell death was due to induction of apoptosis, which occurred earlier and to a greater degree following RL66 and RL118 treatment as opposed to curcumin. Also, RL66 and RL118 were found to be activators of AHR, and a portion of their ability to cause cell death was dependent on this induction. Curcumin was found unable to activate the AHR, and levels of AHR mRNA did not change its effects on cell death. CONCLUSION Piperidone analogs of curcumin exhibited enhanced antitumor effects in vitro as opposed to their parent compound. Even more, this enhanced cell death profile may be partially attributed to the ability of these compounds to activate the AHR. Further study of synthetic curcumin analogs as chemopreventives and chemoadjuncts in CRC is warranted. Also, more generally, the AHR may represent a potential putative target for novel anticancer agents for CRC.

3127 Background: Immune checkpoint inhibitors (ICIs) represent a major advance for treating solid tumors. However, only a minority of patients (pts) benefit from these therapies and markers that predict response have been elusive. Versican (VCAN) is an immunosuppressive proteoglycan in the tumor microenvironment (TME), which releases an immunostimulatory N-terminal fragment versikine (Vkine) when cleaved by ADAMTS proteases. We have demonstrated in colorectal cancers (CRC) that a low VCAN/high Vkine (VCAN proteolytic predominant [VPP]) phenotype correlates with increased tumor-infiltrating CD8+ T lymphocytes (TILs). Here we examine the accumulation of VCAN as a marker of immune exclusion and its proteolysis as a marker of an immune-permissive TME. Methods: Immunohistochemistry for VCAN, Vkine and CD8+ was performed on samples from 1662 pts across breast (BC), CRC, endometrial cancer, pancreatic adenocarcinoma (PDAC), esophageal cancers and neuroendocrine tumors (NETs), across stages of disease (I-IV) and with diverse prior treatments. Stromal intensities of VCAN and Vkine staining quantified in collaboration with blinded surgical pathologists using a 0-3+ scale. 0/1+ were considered “low” for both VCAN and Vkine, whereas 2/3+ were considered “high”. The number of CD8+ TILs were counted using 400x magnification, the equivalent of a high power field (hpf). Results: Across the entire cohort VCAN phenotypes were diverse (VCAN high/Vkine low, 21%; VCAN high/Vkine high, 23%; VCAN low/Vkine low, 29%; VCAN low/Vkine high (VPP), 27%). Consistent with VCAN accumulation as a marker of T cell exclusion, VCAN low cancers had increased TILs compared to VCAN high (4.8 vs 18.3 TILs/hpf, p < 0.001). Low VCAN was identified in 85% esophageal, 79% NET (including small cell lung cancer [SCLC]) 72% endometrial, 47% MSI-H CRCs, 28% triple-negative BC and only 22% MSS CRC, 18% PDAC, 17% ER+ BCs. The VPP subgroup had the highest TILs per hpf across tumors. VPP was identified in 47% of esophageal, 45% endometrial, 41% NETs (including SCLC), 24% MSI-H CRCs, and only 9% MSS CRC, 7% ER+ BCs, 3% triple-negative BCs, and 0% of PDAC (n = 131 PDAC pts). Conclusions: VCAN accumulation correlates with T lymphocyte exclusion, while VCAN proteolysis predicts an immune permissive phenotype. VCAN accumulation and proteolysis are now incorporated into ICI clinical trials as a potential marker of response. Future studies will clarify the role of these biomarkers in predicting benefits of immuno-oncology treatment strategies.

Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response. Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue. Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p&lt;0.001, n=20). VCAN accumulation was increased even in the earliest stages of acinar-to-ductal metaplasia in KPC mice. Areas of intense stromal VCAN staining trended toward reduced CD8+ T cell infiltration (4.9 vs 7.3 cells/high power field (hpf), p=0.3). IHC analysis of human PDAC revealed elevated VCAN accumulation across all stages compared to the normal adjacent tissue (n=231, p&lt;0.001). Areas of high VCAN accumulation demonstrated reduced CD8+ T cells compared to areas of low VCAN (0.6 vs 2.9 cells/hpf, p&lt;0.001). To investigate cell types responsible for enhanced VCAN accumulation in the tumor microenvironment, relative expression (RE) of VCAN was compared in vitro to M0 macrophages. Organotypic cancer spheroids demonstrated increased expression of VCAN from KPC mice (RE=49, n=2) and patient-derived PDAC tissue (RE=14, p=0.01, n=3). M1-polarized BMDMs had increased expression of VCAN (RE=24) compared to M2 BMDMs (RE=8) (p&lt;0.001, n=3). Interestingly, M1 BMDMs cultured in PDAC-conditioned media had reduced RE of M1 markers: TNFα (395 vs 37, p=0.03) and iNOS (24723 vs 4813, p=0.003), and increased M2 markers: Arg-1 (127 versus 1049, p=0.02) and YM-1 (0.5 vs 3, p=0.02). PDAC-conditioned media also reduced VCAN expression of M1 BMDMs (24 vs 9, p=0.02). PSCs derived from human PDAC also demonstrated enhanced RE of VCAN compared to negative controls (RE=68, p=0.017) with no significant change in the presence of PDAC conditioned media (p=0.4). Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Chelsie K. Sievers, Connor J. Maloney, Rosabella T. Pitera, Susan N. Payne, Mitchell G. Depke, Cheri A. Pasch, Linda Clipson, Jillian K. Johnson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican production is driven by both epithelial and stromal cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1904.

<div>Abstract<p><i>PIK3CA</i> mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with <i>PIK3CA</i> mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in <i>APC</i> and <i>PIK3CA</i> mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (−13% and −14%, respectively) compared with an increase of >200% in control (<i>P</i> < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing <i>Pik3ca</i> and <i>Apc</i> mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (<i>P</i> = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in <i>PIK3CA</i> mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.</p></div>