Efficacy of Ibrutinib Monotherapy in Pre-Clinical Mouse Models of Richter Transformation: Ibrutinib Effectively Reduces the Incidence of Richter Transformation but Fails in Treating Transformed Lymphoma, Especially in Primary Lymphoid Tissue
Authors: Chen Shih-Shih , Barrientos Jacqueline , Yan Xiao-Jie , Wirtz Uwe , Szafer-Glusman Edith , Rai Kanti R , Chiorazzi Nicholas 
Published: November 2021

Abstract
Richter's transformation (RT) is a complication of chronic lymphocytic leukemia (CLL) characterized by the development of an aggressive lymphoma, typically diffuse large B cell lymphoma (DLBCL). RT is associated with high expression of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation of non-immunoglobulin genes. Current frontline therapy of CLL, ibrutinib, eliminates AID + CLL cells and alters tissue microenvironment to Th1-biased niches; however approximately 10-20% of CLL patients develop RT upon ibrutinib treatment. In addition, ibrutinib leads to a clinical response in only ~40% of RT patients. Thus, RT remains an important unmet challenge for CLL patients and the mechanism of RT development during ibrutinib treatment is poorly understood.
To determine the contributions of AID and T cells in RT development, and to understand the efficacy of ibrutinib in the treatment of RT, we first created patient-derived xenografts (PDXs) with samples from three RT patients who were refractory to (immuno)chemotherapy but had not previously received ibrutinib. In each case, the B-lymphocytes were larger in size and expressed lower levels of CD5 but higher levels of activation markers compared with the CLL cell counterpart. In PDXs, ibrutinib treatment led to significantly less RT cells in spleen with percentage of inhibition ranging from 28-60% for all three cases. Notably, AID levels were similar in RT cells residing in spleen of ibrutinib-treated and control mice. The numbers of T cells were also similar, although Th1:Th2 ratios were significantly increased by ibrutinib (control vs ibrutinib group: 33% vs 42%, P=0.01). Interestingly, the growth and survival of RT cells located in bone marrow (BM) were not affected by ibrutinib.
We then investigated the efficacy of ibrutinib in the prevention and treatment of RT in a novel strain of TCL-1 mice, TCL1/Igκ-AID mice, that have AID overexpressed solely in B lymphocytes. These mice develop accelerated CLL disease, have shorter overall survival (OS), and bear increased numbers of AID signature mutations in genes that are drivers in CLL and DLBCL.
First we injected primary splenocytes from a TCL1/Igκ-AID mouse (A-31) into wild-type C57BL/6 mice. A week later, mice were treated with vehicle or ibrutinib continuously for 3 weeks (N=5 per group, duplicated experiments). At the end of treatment, RT cells were found in 25% of recipients in the control group and only in 10% of ibrutinib-treated recipients. Ibrutinib-treated animals had overall smaller spleens and lymph nodes (LNs) filled with significantly less CLL or RT cells. Importantly, the balance of Th1/Th2 cytokines was also significantly changed by ibrutinib (Th1/Th2 in control vs ibrutinib: 25% vs 47%, P=0.01). However, again RT cells in BMs of treated and control animals were similar.
We then repeated the experiment to determine overall survival by giving the mice vehicle or ibrutinib continuously. Compared to the TCL1/Igκ-AID (A-31) donors, recipients treated with control vehicle developed more aggressive CLL with shorter OS. However, Ibrutinib did not change the OS; both control and ibrutinib treated recipients had similar OS with all the mice developed and died from RT eventually.
Finally, to determine the efficacy of ibrutinib in the treatment of RT in TCL1/Igκ-AID mice, we sorted A-31-derived RT cells collected from LNs, and injected these into C57BL/6 mice. Mice then received treatment for 3 weeks. Notably, ibrutinib did not change the numbers or sizes of LNs or spleens. The numbers of RT cells at all sites, including BM, remained the same. In both groups, the number of T cells and the Th1:Th2 were not altered by ibrutinib.
In summary, our data suggest that ibrutinib is somewhat effective against primary RT cells in the spleen in PDX models but not at all in the aggressive TCL1/IgK-AID mouse model. This might be because ibrutinib does not reduce AID levels in tissue-resident RT cells. Notably, although RT cells in spleens and LNs respond to ibrutinib, those residing in BM are resistant in both PDX and TCL1/IgK-AID models. This suggests different clones or different ibrutinib-responsiveness of RT cells in primary versus secondary lymphoid tissues. Overall, these results suggest that ibrutinib does not prevent or cure RT. Additionally, TCL1/Igκ-AID mice might provide a model to study the development, prevention, and treatment of RT without the influence of prior chemo- or other therapies.

Disclosures
Wirtz: Abbvie: Current Employment. Szafer-Glusman: AbbVie: Current Employment, Other: Stock or other ownership.