Interrogation of Dysregulated Pathways Enables Precision Medicine in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses after frontline therapy. Although the first-in-class Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has achieved a 68% overall response rate in relapsed/refractory MCL patients, most experience disease progression after ibrutinib treatment. Furthermore, the diverse heterogeneity of molecular alterations in each patient makes improving patient outcome with a uniform regimen extremely challenging. Therefore, identification of effective drug therapies, especially personalized therapeutic strategies are urgently needed. In this study, clinical patient samples were submitted for molecular profiling analysis to identify the dysregulated pathways for each patient. Isolated tumor cells underwent an in vitro drug screen with a panel of clinic drug candidates. Final in vivo efficacy evaluation on the patient derived xenograft (PDX) mouse models could be utilized to predict and validate the clinical response.

Methods: We collected fresh peripheral blood specimens, surgical biopsies, bone marrow aspirates and apheresis samples under established IRB-approved protocols. The extracted tumor RNA was subjected to a CLIA-validated nanoString nCounter analysis to interrogate the dysregulated signaling pathways, and whole-exome sequencing (WES) was conducted to reveal the somatic mutations and DNA copy number alterations. High throughput cell viability assays of 23 clinical drug agents targeting multiple pathways associated with MCL pathogenesis were tested per patient sample using the CellTiter-Glo luminescent assay (Promega). Meanwhile, subcutaneous, intravenous and subrenal injections of the purified patient tumor cells were performed on NSG-mice to create corresponding PDX mouse models and these models were used for in vivo validation of rational therapeutic treatment options for precision medicine.

Results: We collected and submitted 21 clinical patient samples for molecular profiling analysis and screened them through the designed drug panel. We identified correlations between the WES and nanoString nCounter analysis to interrogate the dysregulated pathways for each patient. The PI3K/mTOR signaling, cell cycle regulation, and apoptosis pathways were among the three most markedly enriched pathways in the relapsed MCL samples based on the hallmark gene set analysis. For example, aberrant apoptosis pathway was identified as the predominant cancer hallmark in one of the patients, the Bcl-2 inhibitor venetoclax and MCL-1 inhibitor AZD5991 were chosen as rational therapeutic treatment options. Indeed, both venetoclax and AZD5991 dramatically induced cell death in both primary patient cells and isolated PDX tumor cells ex vivo. Furthermore, administration of venetoclax at 50mg/kg eradicated the tumor growth in the PDX mouse model established from this patient (p<0.001). In another patient, dysregulated cell cycle pathway (E2F targets and G2M checkpoint) was the predominant cancer hallmark and treatment with CDK4/6 inhibitor abemaciclib (75mg/kg) significantly decreased the tumor size in the PDX model derived from this patient (p<0.0001).

Conclusions: Interrogation of dysregulated pathways could be achieved by molecular profiling analysis, and suggest rational treatment options. Follow-up in vitro & in vivo drug efficacy determination facilitates the identification of potential therapeutic options for precision medicine in MCL patient to ultimately improve patient survival outcome.