Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6–12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term Gemcitabine on the tumor microenvironment in order to identify potential therapeutic options for chemo-refractory PDAC. Using a combination of mouse models, primary cell line-derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, Gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring Gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFβ1 biosynthesis. Combined Gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signaling deficiency, Gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that Gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of Gemcitabine-refractory PDAC

New (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones: Synthesis, in vitro cytotoxicity evaluation and apoptosis inducing studies

A new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-one derivatives has been synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines of prostate (PC-3 and DU-145) and breast (BT-549, MDA-MB-231, MCF-7, 4T1), non-small lung (A549) and gastric (HGC) cancer cells along with normal breast epithelial cells (MCF10A). Among the tested compounds, 81 showed significant cytotoxic activity against MDA-MB-231 and 4T1 cancer cells with IC50 values of 3.26 +/- 0.24 mu M and 5.96 +/- 0.67 mu M respectively. The compounds 8f, 8i, 8l and 8o were also screened on normal human breast epithelial cells (MCF10A) and found to be safer with lesser cytotoxicity. The treatment of MDA-MB-231 cells with 81 led to inhibition of cell migration ability through disruption of F-actin protein assembly. The flow-cytometry analysis reveals that the cells arrested in G0/G1 phase of the cell cycle. Further, the compound 81 induced apoptosis of MDA-MB-231 cells was characterized by different staining techniques such as Acridine Orange/Ethidium Bromide (AO/EB), DAPI, annexin V-FITC/PI, Rhodamine-123 and MitoSOX red assay. Western blot studies demonstrated that the compound 81 treatment led to activation of caspase-3, increased expression of cleaved PARP, increased expression of pro-apoptotic Bax and decreased expression of anti-apoptotic Bcl-2 in MDA-MB-231 cancer cells

XP-524 is a dual-BET/EP300 inhibitor that represses oncogenic KRAS and potentiates immune checkpoint inhibition in pancreatic cancer

SignificanceThere are currently no effective treatments for pancreatic ductal adenocarcinoma (PDAC), which displays widespread resistance to chemotherapy, radiation therapy, and immunotherapy. Here, we demonstrate that the multispecificity BET/EP300 inhibitor XP-524 has pronounced single-agent efficacy in vitro, in vivo, and in ex vivo human PDAC slice cultures, functioning in part by attenuating oncogenic KRAS signaling. In vivo XP-524 led to extensive reprogramming of the pancreatic tumor microenvironment, sensitizing murine carcinoma to immune checkpoint inhibition and further extending survival. Given the urgent need for therapeutic approaches in PDAC, the combination of XP-524 and immune checkpoint inhibition warrants additional exploration.</jats:p

Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy

AbstractPancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6–12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine administration on the tumor microenvironment to identify potential therapeutic options for chemorefractory PDAC. Using a combination of mouse models, primary cell line–derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFβ1 biosynthesis. Combined gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signaling deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of gemcitabine-refractory PDAC.Significance:These data suggest that long-term treatment with gemcitabine leads to extensive reprogramming of the pancreatic tumor microenvironment and that patients who progress on gemcitabine-based regimens may benefit from multidrug immunotherapy.See related commentary by Carpenter et al., p. 3070</jats:sec

Overcoming Gemcitabine Resistance in Pancreatic Cancer Using the BCL-XL–Specific Degrader DT2216

Abstract Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro. In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.</jats:p

Figure S5 from Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy

PD-L1/PD-L2 expression is associated with increased immune and TGFβ gene signatures</p

Figure S3 from Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy

Cytotoxic chemotherapy alters the immune surface profile of Capan1 and Bxpc3 tumor cells in vitro</p

Figure S6 from Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy

The combination of Gemcitabine, Galunisertib, and anti-PD-1 restores normal gland architecture and suppresses TGFβ signaling</p

Figure S8 from Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy

Homozygous loss of TP53 enhances MHC Class 1 expression and T-cell infiltration in murine PDAC, yet increased mutational burden predicts poor outcomes</p