The Extracellular Niche and Tumor Microenvironment Enhance KRAS Inhibitor Efficacy in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that lacks effective treatment options, highlighting the need for developing new therapeutic interventions. Here, we assessed the response to pharmacologic inhibition of KRAS, the central oncogenic driver of PDAC. In a panel of PDAC cell lines, inhibition of KRASG12D with MRTX1133 yielded variable efficacy in suppressing cell growth and downstream gene expression programs in 2D cultures. On the basis of CRISPR-Cas9 loss-of-function screens, ITGB1 was identified as a target to enhance the therapeutic response to MRTX1133 by regulating mechanotransduction signaling and YAP/TAZ expression, which was confirmed by gene-specific knockdown and combinatorial drug synergy. Interestingly, MRTX1133 was considerably more efficacious in 3D cell cultures. Moreover, MRTX1133 elicited a pronounced cytostatic effect in vivo and controlled tumor growth in PDAC patient-derived xenografts. In syngeneic models, KRASG12D inhibition led to tumor regression that did not occur in immune-deficient hosts. Digital spatial profiling on tumor tissues indicated that MRTX1133-mediated KRAS inhibition enhanced IFNγ signaling and induced antigen presentation that modulated the tumor microenvironment. Further investigation of the immunologic response using single-cell sequencing and multispectral imaging revealed that tumor regression was associated with suppression of neutrophils and influx of effector CD8+ T cells. Together, these findings demonstrate that both tumor cell-intrinsic and -extrinsic events contribute to response to MRTX1133 and credential KRASG12D inhibition as a promising therapeutic strategy for a large percentage of patients with PDAC. SIGNIFICANCE: Pharmacologic inhibition of KRAS elicits varied responses in pancreatic cancer 2D cell lines, 3D organoid cultures, and xenografts, underscoring the importance of mechanotransduction and the tumor microenvironment in regulating therapeutic responses.

Pharmacologically targeting KRAS G12D in PDAC models: tumor cell intrinsic and extrinsic impact.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease for which new therapeutic interventions are needed. Here we assessed the cellular response to pharmacological KRAS inhibition, which target the central oncogenic factor in PDAC. In a panel of PDAC cell lines, pharmaceutical inhibition of KRAS G12D allele, with MRTX1133 yields variable efficacy in the suppression of cell growth and downstream gene expression programs in 2D culture. CRISPR screens identify new drivers for enhanced therapeutic response that regulate focal adhesion and signaling cascades, which were confirmed by gene specific knockdowns and combinatorial drug synergy. Interestingly, MRTX1133 is considerably more efficacious in the context of 3D cell cultures and in vivo PDAC patient-derived xenografts. In syngeneic models, KRAS G12D inhibition elicits potent tumor regression that did not occur in immune-deficient hosts. Digital spatial profiling on tumor tissues indicates that MRTX1133 activates interferon-γ signaling and induces antigen presentation that modulate the tumor microenvironment. Further investigation on the immunological response using single cell sequencing and multispectral imaging reveals that tumor regression is associated with suppression of neutrophils and influx of effector CD8 + T-cells. Thus, both tumor cell intrinsic and extrinsic events contribute to response and credential KRAS G12D inhibition as promising strategy for a large percentage of PDAC tumors.

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation.

Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.

RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models.

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease.

CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities.

Progression through G1/S phase of the cell cycle is coordinated by cyclin-dependent kinase (CDK) activities. Here, we find that the requirement for different CDK activities and cyclins in driving cancer cell cycles is highly heterogeneous. The differential gene requirements associate with tumor origin and genetic alterations. We define multiple mechanisms for G1/S progression in RB-proficient models, which are CDK4/6 independent and elicit resistance to FDA-approved inhibitors. Conversely, RB-deficient models are intrinsically CDK4/6 independent, but exhibit differential requirements for cyclin E. These dependencies for CDK and cyclins associate with gene expression programs that denote intrinsically different cell-cycle states. Mining therapeutic sensitivities shows that there are reciprocal vulnerabilities associated with RB1 or CCND1 expression versus CCNE1 or CDKN2A. Together, these findings illustrate the complex nature of cancer cell cycles and the relevance for precision therapeutic intervention.

Phase Ib/II Study of Cetuximab plus Pembrolizumab in Patients with Advanced RAS Wild-Type Colorectal Cancer.

PURPOSE: We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC). PATIENTS AND METHODS: In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence. RESULTS: Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9-5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01). CONCLUSIONS: The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.