Patient-derived Cell Line Models Research Articles

EXTH-31. EXPLOITING THE GENETIC DEPENDENCY ON PI3K/MTOR SIGNALING FOR THE TREATMENT OF H3-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract Recurring activating mutations, amplifications and allelic loss of the negative repressors of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling genes are overarching contributors to the poor survival of patients with H3K27-altered diffuse midline gliomas (DMG) diagnosed in the pons (diffuse intrinsic pontine glioma – DIPG), highlighting the clinical potential of therapies that target this axis. Employing a loss-of-function CRISPR-Cas9 gene deletion screen, we identified PIK3CA and MTOR as survival dependences in DMG/DIPG patient derived cell line models (n=36), highlighting the clinical potential of targeting PI3K/Akt/mTOR. The brain-penetrant, PI3K/mTOR inhibitor paxalisib is currently under clinical investigation in DIPG (NCT03696355 and NCT05009992), but dosing limiting toxicities including hyperglycaemia, mucositis and skin irritation limit patient compliance. To exploit this therapeutic vulnerability, we optimized the preclinical use of paxalisib, identifying both precision medicine combination strategies and systemic interventions that reduced side effects and elicited profound tumor control in vivo. Mimicking the patient experience, paxalisib elevated blood glucose levels, promoting the phosphorylation of the insulin receptor (p-INSR) in patient derived DIPG xenograft models. Combining paxalisib with the anti-glycemic drug metformin, synergistically extended the survival of two DIPG xenograft models, decreasing p-Akt, p-mTOR and p-INSR in vivo. Phosphoproteomic profiling of DIPG cells identified paxalisib induced intracellular calcium-regulated PKC signaling. The brain penetrant PKC inhibitor enzastaurin extended the survival of DIPG xenograft models alone, and synergistically combined with paxalisib, enhanced using metformin in three DIPG xenograft models, as well as the immunocompetent H3f3aK27M, PdgfraD842Vmut, Trp53-/- murine brainstem glioma model. Mechanisms of adaptation/plasticity were assessed by ATAC-Seq and quantitative proteomic profiling xenograft tumors refractory to treatment. Here we address the intrinsic neoplastic sequela of DIPG, by combined targeting of PI3K/Akt/mTOR using paxalisib, compensatory PKC signaling using enzastaurin, coupled with strategies to manage treatment-related side-effects and reduced efficacy using metformin; providing the preclinical rationale for the addition of metformin to NCT05009992.

YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.

Abstract 2148: MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines

Abstract Mutations in the extracellular domain (ECD) of EGFR have been reported in colorectal (CRC) patients as a mechanism of acquired resistance to the EGFR-directed antibodies cetuximab and panitumumab. Similar to EGFR intracellular domain mutations observed in patients treated with EGFR kinase inhibitors, these EGFR-ECD mutations inhibit the binding of the antibodies while maintaining EGFR signaling activity. MM-151, an investigational agent consisting of three anti-EGFR IgG1 antibodies, has been recently reported to overcome these mutations in over-expression and patient-derived cell line models. Here, we extend these studies to characterize the mechanisms of action that enable MM-151 to provide robust inhibition of EGFR-ECD mutations beyond that achievable by other monoclonal and oligoclonal EGFR antibodies. First, utilizing a cell-free binding assay with recombinant EGFR-ECD mutants, we found that MM-151, but not the Sym004 combination of two antibodies, maintained oligoclonal binding to all mutants. Second, we performed a panel of 2D and 3D in vitro experiments with over-expression and genome-edited (CRISPR-Cas9) cell lines harboring EGFR-ECD mutations to characterize target engagement, cell signaling, proliferation, and the impact of hetero- versus homozygous expression of EGFR wild type or mutant alleles. We observed that the three mechanisms of action for MM-151- antagonism of both low- and high-affinity EGFR ligands, EGFR down-regulation, and immune-effector activity— were preserved in these models. Third, we performed xenograft experiments to evaluate the behavior of MM-151 and found that MM-151 maintained inhibitory activity in an in vivo context. The results of our multiscale experiments identified the impact of combining three antibodies to overcome the high plasticity of the EGFR extracellular domain and thus reveal the potential for clinical evaluation of MM-151 in both EGFR-naïve and EGFR-refractory CRC populations. Citation Format: Hongfang Wang, Shawn P. Carey, Olga Burenkova, Jian Tang, Nastaran Gerami-Moayed, Sabrina Arena, Alberto Bardelli, Rachel Nering, Jeffrey D. Kearns. MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2148.