Abstract 1071: Targeted therapy evaluation in novel pre-clinical in vitro and in vivo models of Richter transformation
Authors: Fiskus Warren C. , Maiti Abhishek , Saenz Dyana T. , Mill Christopher P. , Khoury Joseph D. , Bhalla Kapil N. 
Published: August 2019

Abstract
Richter Transformation (RT) is the development (in up to 15%) of aggressive DLBCL in patients with antecedent or concurrent CLL. By comparing immunoglobin gene rearrangements, 80% of RT-DLBCL (mostly ABC type) are clonally related (CR) to the underlying CLL, exhibiting poor median survival (MS) of 1 year, whereas 20% of RT-DLBCLs are clonally unrelated (CUR), exhibiting MS of 5 years. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models had prevented pre-clinical testing of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL (CR: RT15 and RT17, CUR: RT5) generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. RT-DLBCL xenografts grew in the bone marrow and spleen, causing marked splenomegaly and requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT5 and RT15 cells showed mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high VAF. Array-CGH showed DNA copy gains or losses in multiple chromosomes including 3, 8, 9, 11, 12, 17 and 18. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities across all super-enhancers/enhancers (SEs/Es), with several SEs/Es exhibiting high scores. ATAC-Seq showed markedly increased signal intensity in RT-DLBCL cells compared to CD34+ normal progenitors, with high peak numbers in specific loci. Western analyses showed that RT15 and RT17 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. RT15 and RT17 cells were more sensitive than RT5 cells to anti-BCL2 venetoclax-induced apoptosis (p &lt; 0.01). RT15 and RT17, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT15 and RT17 cells to ibrutinib-induced apoptosis. RT15 and RT17 cells were also less sensitive than RT5 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices &lt; 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 dose-dependently induced apoptosis in RT-DLBCL cells which was associated with depletion of c-Myc, Bcl-xL and MCL1 protein levels. These findings highlight the activity and support further evaluation of in vitro and in vivo 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.
Citation Format: Warren C. Fiskus, Abhishek Maiti, Dyana T. Saenz, Christopher P. Mill, Joseph D. Khoury, Kapil N. Bhalla. Targeted therapy evaluation in novel pre-clinical in vitro and in vivo models of Richter transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1071.