KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+; Trp53LSL-R172H/+; p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, Cdk6, and Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies. Significance: Acquired resistance may limit the impact of KRAS inhibition in patients with PDAC. Using clinical samples and multiple preclinical models, we define heterogeneous genetic and non-genetic mechanisms of resistance to KRAS inhibition that may guide combination therapy approaches to improve the efficacy and durability of these promising therapies for patients. See related commentary by Marasco and Misale, p. 2018

From the development of self-aggregating, scaffold-free multicellular spheroids to the inclusion of scaffold systems, 3D models have progressively increased in complexity to better mimic native tissues. The inclusion of a third dimension in cancer models allows researchers to zoom out from a significant but limited cancer cell research approach to a wider investigation of the tumor microenvironment. This model can include multiple cell types and many elements from the extracellular matrix (ECM), which provides mechanical support for the tissue, mediates cell-microenvironment interactions, and plays a key role in cancer cell invasion. Both biochemical and biophysical signals from the extracellular space strongly influence cell fate, the epigenetic landscape, and gene expression. Specifically, a detailed mechanistic understanding of tumor cell-ECM interactions, especially during cancer invasion, is lacking. In this review, we focus on the latest achievements in the study of ECM biomechanics and mechanosensing in cancer on 3D scaffold-based and scaffold-free models, focusing on each platform’s level of complexity, up-to-date mechanical tests performed, limitations, and potential for further improvements.

The mechanical homeostasis of tissues can be altered in response to trauma or disease, such as cancer, resulting in altered mechanotransduction pathways that have been shown to impact tumor development, progression, and the efficacy of therapeutic approaches. Specifically, ovarian cancer progression is parallel to an increase in tissue stiffness and fibrosis. With in vivo models proving difficult to study, tying tissue mechanics to altered cellular and molecular properties necessitate advanced, tunable, in vitro 3D models able to mimic normal and tumor mechanic features. First, we characterized normal human ovary and high-grade serous (HGSC) ovarian cancer tissue stiffness to precisely mimic their mechanical features on collagen I-based sponge scaffolds, soft (NS) and stiff (MS), respectively. We utilized three ovarian cancer cell lines (OVCAR-3, Caov-3, and SKOV3) to evaluate changes in viability, morphology, proliferation, and sensitivity to doxorubicin and liposomal doxorubicin treatment in response to a mechanically different microenvironment. High substrate stiffness promoted the proliferation of Caov-3 and SKOV3 cells without changing their morphology, and upregulated mechanosensors YAP/TAZ only in SKOV3 cells. After 7 days in culture, both OVCAR3 and SKOV3 decreased the MS scaffold storage modulus (stiffness), suggesting a link between cell proliferation and the softening of the matrix. Finally, high matrix stiffness resulted in higher OVCAR-3 and SKOV3 cell cytotoxicity in response to doxorubicin. This study demonstrates the promise of biomimetic porous scaffolds for effective inclusion of mechanical parameters in 3D cancer modeling. Furthermore, this work establishes the use of porous scaffolds for studying ovarian cancer cells response to mechanical changes in the microenvironment and as a meaningful platform from which to investigate chemoresistance and drug response.

Patients with BRAF-mutant melanoma show substantial responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically “cold” after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines as strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining chemokine signaling could impair MRD maintenance through increased recruitment of effector T cells. We found that intratumoral administration of recombinant Cxcl9 (rCxcl9), either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete pathologic responses after microparticle-delivered rCxcl9 combined with BRAF and MEK inhibition. Our experiments constitute proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of first-line treatment methods.

Mutations in KRAS are a dominant driver of pancreatic ductal adenocarcinoma (PDAC), with over 40% of PDAC patients presenting with KRAS ^G12D mutations. The recent development of small molecule inhibitors targeting KRAS ^G12D has enabled targeting of mutant KRAS signaling and suppression of PDAC; however, the contribution of the tumor microenvironment (TME) to the sustained therapeutic efficacy of KRAS ^G12D inhibition and mechanism/s of resistance to KRAS ^G12D suppression remain to be elucidated. Here, we employed spatial transcriptomics, single cell RNA sequencing, and CODEX-based spatial proteomics to evaluate cancer cell intrinsic and extrinsic responses to KRAS ^G12D inhibition with MRTX1133. While KRAS ^G12D inhibition initially increases CD11c ⁺ cells with impactful T cell infiltration within proximity to cancer cells, long-term treatment with MRTX1133 resulted in reversal of the immune responses leading to KRAS ^G12D therapy resistance promoted by CDK8, a multiprotein mediator complex associated kinase. CDK8 imparts resistance in part through induction of downstream CXCL2 chemokine secretion, inhibition of FAS expression, and remodeling of the TME to promote immune evasion. Targeting CDK8 by itself and in combination with αCTLA-4 immunotherapy overcomes resistance to KRAS ^G12D inhibition with prolonged survival with translational implications.