Cytotoxic chemotherapy potentiates the immune response and efficacy of combination CXCR4/PD-1 inhibition in models of pancreatic ductal adenocarcinoma.

Purpose: The CXCL12-CXCR4 chemokine axis plays a significant role in modulating T-cell infiltration into the pancreatic tumor microenvironment. Despite promising preclinical findings, clinical trials combining inhibitors of CXCR4 (AMD3100/BL-8040) and anti-programmed death 1/ligand1 (anti-PD1/PD-L1) have failed to improve outcomes. Experimental Design: We utilized a novel ex vivo autologous patient-derived immune/organoid (PDIO) co-culture system using human peripheral blood mononuclear cells and patient derived tumor organoids, and in vivo the autochthonous LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) pancreatic cancer mouse model to interrogate the effects of either monotherapy or all combinations of gemcitabine, AMD3100, and anit-PD1 on CD8+ T cell activation and survival. Results: We demonstrate that disruption of the CXCL12-CXCR4 axis using AMD3100 leads to increased migration and activation of CD8+ T-cells. In addition, when combined with the cytotoxic chemotherapy gemcitabine, CXCR4 inhibition further potentiated CD8+ T-cell activation. We next tested the combination of gemcitabine, CXCR4 inhibition, and anti-PD1 in the KPC pancreatic cancer mouse model and demonstrate that this combination markedly impacted the tumor immune microenvironment by increasing infiltration of natural killer cells, the ratio of CD8+ to regulatory T-cells, and tumor cell death while decreasing tumor cell proliferation. Moreover, this combination extended survival in KPC mice. Conclusions: These findings suggest that combining gemcitabine with CXCR4 inhibiting agents and anti-PD1 therapy controls tumor growth by reducing immunosuppression and potentiating immune cell activation and therefore may represent a novel approach to treating pancreatic cancer.

Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer.

To identify novel drivers of malignancy in pancreatic ductal adenocarcinoma (PDAC), we employed regulatory network analysis, which calculates the activity of transcription factors and other regulatory proteins based on the integrated expression of their positive and negative target genes. We generated a regulatory network for the malignant epithelial cells of human PDAC using gene expression data from a set of 197 laser capture microdissected human PDAC samples and 45 low-grade precursors, for which we had matched histopathological, clinical, and epidemiological annotation. We then identified the most highly activated and repressed regulatory proteins (e.g. master regulators or MRs) associated with four malignancy phenotypes: precursors vs. PDAC (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Integrating across these phenotypes, the top MR of PDAC malignancy was found to be BMAL2, a member of the PAS family of bHLH transcription factors. Although the canonical function of BMAL2 is linked to the circadian rhythm protein CLOCK, annotation of BMAL2 target genes highlighted a potential role in hypoxia response. We previously demonstrated that PDAC is hypovascularized and hypoperfused, and here show that PDAC from the genetically engineered KPC model exists in a state of extreme hypoxia, with a partial oxygen pressure of <1mmHg. Given the close homology of BMAL2 to HIF1ß (ARNT) and its potential to heterodimerize with HIF1A and HIF2A, we investigated whether BMAL2 plays a role in the hypoxic response of PDAC. Indeed, BMAL2 controlled numerous hypoxia response genes and could be inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its association with RAS activity. Knockout of BMAL2 in four human PDAC cell lines led to defects in growth and invasion in the setting of hypoxia. Strikingly, BMAL2 null cells failed to induce glycolysis upon exposure to severe hypoxia and this was associated with a loss of expression of the glycolytic enzyme LDHA. Moreover, HIF1A was no longer stabilized under hypoxia in BMAL2 knockout cells. By contrast, HIF2A was hyper-stabilized under hypoxia, indicating a dysregulation of hypoxia metabolism in response to BMAL2 loss. We conclude that BMAL2 is a master regulator of hypoxic metabolism in PDAC, serving as a molecular switch between the disparate metabolic roles of HIF1A- and HIF2A-dependent hypoxia responses.

Hypothermia revisited: Impact of ischaemic duration and between experiment variability.

To assess the true effect of novel therapies for ischaemic stroke, a positive control that can validate the experimental model and design is vital. Hypothermia may be a good candidate for such a positive control, given the convincing body of evidence from animal models of ischaemic stroke. Taking conditions under which substantial efficacy had been seen in a meta-analysis of hypothermia for focal ischaemia in animal models, we undertook three randomised and blinded studies examining the effect of hypothermia induced immediately following the onset of middle cerebral artery occlusion on infarct volume in rats (n = 15, 23, 264). Hypothermia to a depth of 33℃ and maintained for 130 min significantly reduced infarct volume compared to normothermia treatment (by 27-63%) and depended on ischaemic duration (F(3,244) = 21.242, p < 0.05). However, the protective effect varied across experiments with differences in both the size of the infarct observed in normothermic controls and the time to reach target temperature. Our results highlight the need for sample size and power calculations to take into account variations between individual experiments requiring induction of focal ischaemia.