Background: Stromal hyaluronic acid (HA) poses a physical barrier and protects tumor cells from immune surveillance. Stroma targeting with pegylated human recombinant PH20 hyaluronidase (PEGPH20) demonstrated improved infiltration of cytotoxic T-lymphocytes and delivery of chemotherapy and PD1/PD-L1 antibodies in tumor models. This multicenter phase II study of PEGPH20 plus pembrolizumab evaluated the efficacy, safety and immune and stromal biomarkers in patients with HA-high refractory metastatic pancreatic ductal adenocarcinoma (mPDA). Patients and Methods: Patients were treated with PEGPH20 3 µg/kg IV weekly and pembrolizumab 200 mg IV in 3-week cycles. Tumor and blood samples were collected at baseline and on-study for biomarker analyses. Results: Between May and November 2019, 38 patients were screened and 8 treated, with median age 68 years (range 60-73) and median two (range 1-4) prior therapies. The study was closed to accrual early by pharmaceutical sponsor. Treatment was well tolerated, with expected grade 1/2 musculoskeletal toxicities. Best response was stable disease in 2 of 7 evaluable patients (29%). Median overall and progression-free survival were 7.2 months (95% CI 1.2-11.8) and 1.5 months (95% CI 0.9-4.4), respectively. Prolonged survival (range 10.2-27.6 months) occurred in patients treated with subsequent chemotherapy. Higher baseline tumor T cell receptor (TCR) clonality correlated with longer survival. Conclusions: Pembrolizumab with PEGPH20 was safe but did not have significant efficacy in refractory HA-high metastatic PDA.

Supplementary Figure S2. Demographic data for analyzed cohort. A) Age distribution for TMA and Ochsner cohorts. B) Race distribution for TMA and Ochsner cohorts. C) Proportions of disease by stage (left) and resulting survival (right) predicts survival in TMA cohort (AJCC 8th edition).

Supplementary Table S3. HMGA2 vs GATA6 Differential Genes.

Supplementary Figure S6. GATA6 expression predicts chemotherapy response to GnP but not mFFX. A) GATA6low metastatic tumors have significantly worse overall survival when treated with combination GnP first-line chemotherapy. B) GATA6 status does not predict overall survival when patients receive first line mFFX chemotherapy.

Purpose: The purpose of this study was to establish HMGA2 as a marker of basal-like disease in pancreatic ductal adenocarcinoma (PDAC) and explore its use as a biomarker for prognosis and treatment resistance.Experimental Design: We identified high-mobility group A2 (HMGA2) protein expression in basal PDAC cells in a single-cell RNA sequencing (RNA-seq) atlas of 172 patient samples. We then analyzed HMGA2 expression, along with expression of the classic marker GATA-binding factor 6 (GATA6), in a cohort of 580 PDAC samples with multiplex IHC. We further supplemented these data with an additional 30 diverse patient samples and multiple independent single-cell RNA-seq databases.Results: We found that expression of HMGA2, but not previously described basal markers cytokeratins 5 or 17, predicted overall survival in our cohort. Combining HMGA2 and GATA6 statuses allowed for the identification of two key study groups: an HMGA2+/GATA6- cohort with worse survival, low tumor-infiltrating CD8+ T cells, increased FAP+ fibroblasts, and poorer response to gemcitabine-based chemotherapies (n = 94, median survival = 11.2 months after surgery) and an HMGA2-/GATA6+ cohort with improved survival, increased CD8+ T-cell infiltrate, decreased FAP+ fibroblasts, and improved survival with gemcitabine-based chemotherapy (n = 198, median survival = 21.7 months after surgery). HMGA2 was also prognostic for overall survival in RNA-seq from an independent cohort.Conclusions: IHC stratification of primary tumors by HMGA2 and GATA6 statuses in pancreatic cancer is associated with differential outcomes, survival following chemotherapy, and tumor microenvironments. As a nuclear marker for basal disease, HMGA2 complements GATA6 to identify disease subtypes in PDAC.

Supplementary Figure S5. Representative CD8+ and FAP+ scoring. CD8 (top) and FAP (bottom) were scored on a scale of 0 (least staining) to 3 (most staining) following the representative scale.

Supplementary Figure S3. Consort Diagram and Mixed Populations. A) Consort diagram of analyzed samples. For survival analyses, all patients with intact cores (n = 491) were assessed. For treatment response analyses, patients were further subdivided by type of chemotherapy received, and neoadjuvant-treated samples were considered separately (n = 51). B) Mixed (HMGA2+ GATA6+) and double negative (HMGA2- GATA6-) populations show intermediate survival between basal and classical tumors.

Supplementary Table S5. Representativeness of Study Participants.

Supplementary Figure S1. Example images of IHC of FFPE samples. A) HMGA2 positive glands. B) GATA6 positive glands. Scale bars = 50 μm.

Supplementary Figure S4. CK5 and CK17 do not predict treatment response or time to recurrence. A) Combining CK5 and GATA6 IHC does not predict overall survival. B) CK17 high, GATA6 low tumors display improved survival compared to CK17 low, GATA6 high tumors. C) Kaplan-Meier survival curves for CK5 positive and negative tumors treated with first-line gemcitabine chemotherapy. D) Kaplan-Meier survival curves for CK17 positive and negative tumors treated with first-line gemcitabine chemotherapy. E) CK5 status does not predict time to recurrence. F) CK17 positive tumors show longer time to recurrence than CK17 negative tumors, the opposite phenotype expected from the basal subtype.

Pancreatic ductal adenocarcinoma (PDA) is a deadly malignancy with limited effective therapies. Adoptive cell therapy (ACT) is a promising treatment modality for patients with solid tumors but has been limited by the highly fibroinflammatory and immunosuppressive tumor microenvironment (TME). Transforming growth factor-β (TGFβ) participates in the inordinately suppressive TME in PDA. Here, we test the impact of selective Tgfbr2 deletion using CRISPR/Cas9 or genetic approaches in mesothelin (Msln)-specific T cell receptor (TCR) engineered T cells during ACT of PDA. Abrogating TGFβ signaling augmented TCR-engineered T cell accumulation in autochthonous and orthotopic PDA models and promoted terminal effector T cells, although this largely required inclusion of a vaccine at the time of T cell transfer. While loss of Tgfbr2 impaired CD103 upregulation, it only modestly impaired donor T cell central, tissue resident, or Tcf1+Slamf6+ stem-like memory T cell formation. These attributes ultimately result in heightened functional capacity and delayed tumor growth. Unexpectedly, however, most tumor-infiltrating engineered T cells failed to differentiate into PD-1+Lag3+ exhausted T cells (TEX) regardless of TGFβR2 expression and despite abundant Msln protein expression by PDA cells. Forcing Msln epitope processing in KPC tumor cells promoted donor T cell accumulation, acquisition of PD-1 and Lag3, increased IFNγ production by TCR-engineered T cells refractory to TGFβ and bypassed the vaccine requirement for therapeutic efficacy. Thus, promoting increased antigen processing/presentation by tumor cells while abrogating Tgfbr2 in engineered T cells can sustain donor T cell function in the suppressive TME and enhance the therapeutic efficacy of ACT. Our study supports pursuit of strategies that modulate tumor intrinsic antigen processing while relieving T cell suppression to safely promote the antitumor activity of TCR-engineered T cells. ### Competing Interest Statement I.M.S. serves on the scientific advisory boards for Luminary Therapeutics and Immunogenesis, has a sponsored research project with Bonum therapeutics, and has patents in human T cellular engineering constructs and the TRex mouse model. P.P.P. is on the scientific advisory board for Incindia Therapeutics. P.D.G. is a cofounder of Affini-T Therapeutics and Juno Therapeutics, is on the scientific advisory boards of Affini-T Therapeutics, Immunoscape, RAPT, Earli, Catalio, Nextech, Metagenomi, and Elpiscience, and has received research support from Affini-T, Juno and Lonza. The remaining authors declare no competing interests. National Institutes of Health, https://ror.org/01cwqze88, T32CA009138, T32AI007313, R01CA255039, R01CA249393, U54CA268069, P01CA254849 United States Department of Defense, https://ror.org/0447fe631, W81XWH2110525 American Association For Cancer Research, 19-35STRO American Cancer Society, https://ror.org/02e463172, RSG-21-102-01-IBCD

Supplementary Table S2. mIHC Panel.

Abstract Background: Psychological distress is a common reaction to a cancer diagnosis and its treatment. Distress impacts quality of life, adherence to treatment, decision making and survival. Patients with pancreatic adenocarcinoma (PDAC) experience exceptionally high rates of psychological distress, most commonly depression, anxiety and suicidality. Psilocybin, a serotonergic psychedelic which is the active component in “magic mushrooms,” is a promising treatment for several mental health disorders. Prior studies in patients with cancer have combined a single dose of psilocybin with preparation and integration counseling and have shown significant, rapid and sustained improvement in depression, anxiety and quality of life. These studies have primarily been conducted in psychiatry or neuroscience clinics in heterogenous groups of patients, many of whom had completed cancer treatment. This study will evaluate the feasibility and safety of psilocybin-assisted therapy delivered in the outpatient palliative care clinic in patients with advanced PDAC who are receiving chemotherapy. Methods: This is a single center, open label pilot study of a psilocybin-assisted therapy integrated with palliative care in patients with advanced PDAC. The primary endpoints are the feasibility and safety of this strategy when integrated into routine palliative care. Secondary endpoints include short term changes in depression, anxiety and demoralization and changes in neuronal activity by functional MRI (fMRI). After 2-4 outpatient preparation sessions with a palliative medicine physician (PMP), the subject will take psilocybin (25mg capsule) at an individual session supported by a PMP and a trained mental health professional. Up to 3 integration sessions with a PMP will follow. Patient-reported measures of psychological distress and quality of life will be assessed at baseline, after the psilocybin dose and periodically during follow-up. Neuronal changes will be measured using fMRI before and after the psilocybin dose. Eligible patients are adults (ECOG 0-3) with advanced PDAC with adequate organ function. Patients with psychiatric conditions which preclude safe cessation of psychotropic drugs or requires hospitalization, a personal or family history of schizophrenia, psychotic disorder or bipolar disorder, who have a high risk of suicide or have serious or uncontrolled hypertension or cardiovascular disease are ineligible. Given the exploratory nature of the study, a formal statistical assessment of the sample size was not conducted. Twelve evaluable subjects will provide sufficient power to detect large effect sizes (Cohen’s d1.0) in the secondary endpoints. Wilcoxon signed rank test will determine the reduction in distress scores before and after the psilocybin session. Enrollment began in June 2023; two subjects have enrolled. The study is funded by the Jim Young Pancreatic Cancer Research Memorial Fund. Psilocybin is provided by Usona. ClinicalTrials.gov Identifier: NCT05220046 Citation Format: Kelsey A. Klute, Lou Lukas, Soonjo Hwang, Yeongjin Gwon, Mridula Krishnan, Jean Grem, Sunil Hingorani, Jody Koenig Kellas. A pilot study of palliadelic treatment with psilocybin to reduce psychological distress and improve quality of life in patients with advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C001.

Abstract Pancreatic ductal adenocarcinoma (PDA) is classified into two distinct subtypes, classical and basal, with the basal subtype predicting worse survival in patients. We hope that with a deeper understanding of the biology that drives these subtypes, we can identify novel subtype-specific therapies. We recently described a novel finding that basal PDAs are uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9. Additionally, we identified that Sirtuin 6 histone deacetylase (SIRT6) expression levels correlate with PDA subtype, showing that basal PDA is low in SIRT6 expression and classical PDA is high through analysis of hundreds of human PDA tumors. Furthermore, we uncovered an important mechanism by which SIRT6 regulation of the integrated stress response (ISR) can control sensitivity of PDA to CDK7/9 inhibitors. By using a variety of in vitro drug assays, genetic manipulation experiments, immunofluorescence, cycloheximide chase experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDA, we were able to show that SIRT6 regulates activating transcription factor 4 (ATF4) protein stability. Therefore, SIRT6 high/classical PDA has a constitutively active ISR, enabling it to recover and survive the stress of CDK7/9 transcriptional inhibition. On the other hand, basal PDA has low SIRT6 which promotes ATF4 protein instability and a nonfunctional ISR, therefore CDK7/9 transcription inhibition leads to apoptosis. Furthermore, we found that similar to intrinsic resistance, acquired resistance to CDK7/9 inhibitors occurs through the loss of basal state drivers and that restoration of these genes re-sensitizes cells to CDK7/9 inhibition. Importantly, this work highlights the role of chromatin modifiers in regulating PDA subtypes and sensitivity to CDK7/9 inhibitors. PDA subtypes have very distinct chromatin landscapes. We have found that classical PDA maintains its epithelial cell state through chromatin regulators like the SETDB1 heterochromatin complex and more specifically the KRAB-Zinc Finger protein ZNF274. Loss or gain of ZNF274 can induce a cell state change in which classical PDA loses its epithelial characteristics and becomes more mesenchymal in nature. Excitingly, this sensitizes cells to CDK7/9 inhibition. Thus, our work demonstrates the importance of cell state and subtype in therapeutic response to CDK7/9 inhibitors in pancreatic cancer. Citation Format: Sita Kugel, Nithya Kartha, Jessica E. Gianopulos, Zach Schrank, Sarah M. Cavender, Stephanie Dobersch, Notta Faiyaz, David H. Price, Andrew C. Hsieh, Sunil R. Hingorani. Targeting CDK7/9 in basal pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR12.

Supplementary Figure S1. A multispectral image of an in vivo murine subcutaneous pancreatic tumor treated with pHIFU with varying levels of peak-rarefactional pressure.

Supplementary Figure Legends. Legends for Supplementary Figures S1-S2.

S1. TLS composition and immunological features of resected PDA. S2. Gating strategy for analyzing CD244 (2B4) and Tim3 expression on CD8 T cells in blood of normal individuals (Nl), in blood of PDA patients (PDA blood) and in tumors. S3. Representative human PDA section stained with the multiplex immunohistochemical panel. S4. Representative images of human PDA from the multiplex without (top row, A and B) and with (bottom, C and D), the stromal vs. tumor regions demarcated and cell numbers quantified. S5. PD-L1+ stromal cells in PDA include CD163+ macrophages (Figure 6A) as well as an additional CD163- cell subset shown here. Supplementary Table 1. Antibodies used for tissue multiplex in Figure 6. Supplementary Table 2. Patient characteristics. Supplementary Table 3. TCRβ CDR3 sequence data in blood and tumors from PDA patients.

Figure S2 - PDF file 2023K, IHC images for HIF-1 and SHH in patient matched tumor TMA cores