Stem and progenitor cell fate transitions constitute key decision points in organismal development that enable access to a developmental path or actively preclude others. Using the hematopoietic system, we analyzed the relative importance of cell fate–promoting mechanisms versus negating fate-suppressing mechanisms to engineer progenitor cells with multilineage differentiation potential. Deletion of the murine Gata2−77 enhancer, with a human equivalent that causes leukemia, downregulates the transcription factor GATA2 and blocks progenitor differentiation into erythrocytes, megakaryocytes, basophils, and granulocytes, but not macrophages. Using multiomics and single-cell analyses, we demonstrated that the enhancer orchestrates a balance between pro- and anti-fate circuitry in single cells. By increasing GATA2 expression, the enhancer instigates a fate-promoting mechanism while abrogating an innate immunity–linked, fate-suppressing mechanism. During embryogenesis, the suppressing mechanism dominated in enhancer mutant progenitors, thus yielding progenitors with a predominant monocytic differentiation potential. Coordinating fate-promoting and -suppressing circuits therefore averts deconstruction of a multifate system into a monopotent system and maintains critical progenitor heterogeneity and functionality.

Conclusions: FOLFOX treatment impacts the phenotype and function of TILs making them more responsive to checkpoint blockade. This study highlights the importance of combining chemotherapy and ICB to optimize treatment efficacy in patients with colorectal cancer.

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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.

Introduction: The purpose of this study is to correlate T cell subset abundance with the accumulation and proteolysis of versican (VCAN) in colorectal cancer (CRC). Background: In CRC, VCAN accumulation is associated with the exclusion of CD8+ tumor infiltrating lymphocytes (TILs) and its proteolysis, into versikine (Vkine), correlates with increased TIL abundance. Here we additionally investigate for changes in CD4+ T cells, T-regulatory cells (Tregs), and T-helper-1 cells (TH1s) in association with the presence of VCAN and Vkine. Methods: VCAN, Vkine, TIL, and T cell abundance were assessed via immunohistochemistry or immunofluorescence in 455 human CRC samples. VCAN and Vkine were scored using an intensity binning system ranging from 0-3+ and T cells were counted per high power field (HPF). Tumors were designated as high (2 or 3+) or low (0 or 1) for both VCAN and Vkine. Cores designated VCAN low and Vkine high are considered VCAN proteolytic predominant (VPP) and all other combinations are considered VCAN proteolytic weak (VPW). CD4 subsets were visualized by colocalizing CD4 with FOXP3 for Tregs and Tbet for TH1s using dual immunofluorescence. Findings: High VCAN accumulation correlated with low TIL abundance. VCAN high cancers had 6 CD8+ TILs/HPF while VCAN low cases had 20 CD8+ TILs/HPF (p&lt;0.001). VCAN proteolysis correlated with increased CD8+ TIL abundance. VPP samples had 46 CD8+ TILs/HPF and VPW ones had 6 CD8+ TILs/HPF (p&lt;0.001). When evaluating total CD4+ TIL abundance, no association was found with either VCAN accumulation or proteolysis. When evaluating CD4 subset abundance, VCAN accumulation had no predictive value for TH1 numbers, but VCAN proteolysis correlated with increased TH1 abundance. VPP cancers had 25 TH1s/HPF and VPW cancers had 9 TH1s/HPF (p=0.02). Neither VCAN accumulation nor proteolysis was predictive of Treg abundance. Accordingly, VCAN high cores had a lower TIL: Treg ratio than VCAN low cores, with VCAN high samples having a CD8+ TIL: Treg ratio of 0.4:1 and VCAN low samples having a ratio of 1:1 (p&lt;0.001). VPP cases showed an elevated TIL: Treg ratio compared to VPW CRCs, with VPP cancers having a CD8+ TIL: Treg ratio of 2:1 and VPW having a ratio of 0.4:1 (p=0.001). Conclusions: Here we demonstrate that VCAN accumulation within the CRC TME is associated with fewer CD8+ TILs and a lower TIL: Treg ratio. Conversely, high levels of VCAN proteolysis correlated with increased CD8+ TIL and TH1 abundance, and a greater TIL: Treg ratio. This indicates that VCAN accumulation and proteolysis differentially predict the abundance of different T cell subtypes and polarization states which could be important for immunotherapy responses. Citation Format: Sean G Kraus, Anna Field, Derek Buckalew, Cheri Pasch, Dustin Deming. Versican accumulation and proteolysis differentially predict T cell subset abundance within the colorectal cancer tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B34.