Efficacy of Targeted Therapy in Novel Pre-Clinical in Vitro and In Vivo Models of Richter Transformation-Diffuse Large B-Cell Lymphoma (RT-DLBCL)
Authors: Karkhanis Vrajesh , Fiskus Warren , Mill Christopher Peter , Maiti Abhishek , Lara Bernardo H , Maher James V , Bhalla Isean , Shao Nancy , Green Michael R , Khoury Joseph D. , Bhalla Kapil N. 
Published: November 2019

Richter Transformation (RT) is defined as the development of aggressive DLBCL (mostly ABC-type) in up to ~15% of patients with antecedent or concurrent diagnosis of CLL. Based on the comparison of immunoglobin gene rearrangements, approximately 80% of RT-DLBCL arise due to a direct clonal evolution of the underlying CLL clone, i.e., clonally related (CLR) RT-DLBCL, which exhibit poor median survival (MS) of one year. Approximately 20% of RT-DLBCLs are clonally unrelated (CUR) to the underlying CLL, arising most likely due to branched clonal evolution from a common pre-CLL progenitor. CUR RT-DLBCLs exhibit a better MS of 5 years. Although chemo-immunotherapy and treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or anti-apoptotic BCL2 inhibitor venetoclax can induce remissions, they fail to induce prolonged disease-free survival in RT-DLBCL. Majority of patients relapse with therapy-refractory disease. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models has prevented pre-clinical testing and development of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL. Based on immunoglobulin heavy chain (IGH) clonality testing by NGS, the RT-DLBCL RT17 was CLR, RT15 was CUR and RT5 was of indeterminate clonality. The PDXs were generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. The RT-DLBCL PDXs grew in the bone marrow and spleen, causing marked splenomegaly, requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL PDX cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT17, RT15, and RT5 cells showed large numbers of genetic mutations, including mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high variant allelic frequencies. Array-CGH showed DNA copy gains or losses in multiple chromosomes, including 3, 8, 9, 11, 12, 17 and 18. A 5'-MYC amplification was detected by FISH analysis in RT5 DLBCL cells. ATAC-Seq showed increased signal intensity representing increased chromatin accessibility in the RT-DLBCL cells compared to CD34+ normal progenitors. High peak numbers were detected in specific loci, including TCF4, PAX5, IRF4, MYB, MYC, BCL2L1 and BCL-2. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities at super-enhancers/enhancers (SEs/Es), including those of TCF4, PAX5, IRF4, BCL2 (RT17 and RT15) and MYC (RT5). Western analyses showed that all three RT-DLBCL PDX cells expressed TCF4, c-Myc, and BRD4, with highest expression in RT5 cells. Accordingly, RT5 cells were more sensitive than cells RT17 and RT15 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. This was associated with greater, OTX015-mediated, depletion of c-Myc in RT5 cells. RT17 and RT15 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. Consistent with this, RT17 and RT15 cells were significantly more sensitive than RT5 cells to venetoclax-induced apoptosis (p < 0.01). RT17 and RT15, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT17 and RT15 cells to ibrutinib-induced apoptosis (p < 0.001). However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices < 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of all RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 also dose-dependently induced apoptosis in RT-DLBCL cells associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. These findings highlight the activity and support further in vitro and in vivo evaluation of BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically-profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL.

Disclosures
Maiti: Celgene: Other: research funding. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding.