Profiling drug sensitivity in CLL B cells after BTK inhibitor progression using a novel drug panel

Abstract

Abstract Introduction: Although current clinical treatment approaches targeting Bruton Tyrosine Kinase (BTK) and BCL2, either as monotherapy or in combination are highly effective in chronic lymphocytic leukemia (CLL), CLL remains incurable. Identifying novel drugs that are potent and effective in the relapsed/refractory (RR) setting remains an unmet need. Methods: Utilizing the CellTiter-Glo 2.0 Cell Viability Assay, we have developed a preclinical drug screening platform which allows conducting drug sensitivity profiling of primary peripheral blood mononuclear cells (PBMCs) collected from CLL patients to a panel of drugs, in the presence and/or absence of bone marrow stromal cells (BMSCs). This unique system mimics the in vivo stromal microenvironment in CLL and enables testing of multiple drug responses in primary patient samples, particularly when leukemic cell numbers are low. In this study, we employed this high throughput drug screening platform to profile drug sensitivity in CLL B cells after disease progression on a BTK inhibitor (BTKi) to a panel of 12 drugs, including inhibitors of BCL2 (venetoclax and LP-118 [Newave]), AXL (TP-0903), histone deacetylase (panobinostat and belinostat), p53 (eprenetapopt and idasanutlin), proteasome (carfizomib) and Type III Receptor Tyrosine Kinase (crenolanib), next generation BTKi (LP-168 [Newave]), as well as a novel anti-cancer drug transcriptional chemical inducer of proximity (TCIP1) and the natural green tea extract (GTE) product Sunphenon 90D (Taiyo International). After 24-hour drug exposure, leukemic cell viability was measured, killing curves and IC50 levels generated and calculated by nonlinear curve regression in GraphPad Prism. Results: Using the Mayo Clinic CLL Database and the Mayo Clinic CLL Tissue Bank, we identified 47 patients with RR CLL, all of whom experienced disease progressionon a BTKi (35 ibrutinib +/- CD20 antibody, 10 acalabrutinib +/- CD20 antibody, 2 other). Median age at BTKi initiation was 64 years (range, 41-82), and 36 (77%) were male. IGHV status was unmutated in 44 (94%) patients, FISH at diagnosis showed del17p in 11 (27%), del11q in 6 (15%), +12 in 5 (12%), del13q in 12 (30%), and not detected in 6 (15%) patients. TP53 mutation was seen in 13 (32%) patients at the start of BTKi therapy. Median number of lines of CLL therapy prior to start of BTKi was 2 (range, 0–8); 13 patients received BTKi as their first-line therapy. Our drug screening results showed definite responses to all tested drugs based on dose range generated killing curves and IC50 levels for 30 RR CLL patient samples, though results varied. Among 12 drugs tested, LP-118 (0.1nM), venetoclax (2.6nM), carfizomib (2.2nM), TP-0903 (116.2nM), crenolanib (1769nM), and TCIP1 (1805nM) demonstrated the highest killing effects, while the remaining generated killing but at higher doses: belinostat (6800nM), eprenetapopt (4500nM), idasanutlin (4900nM), LP-168 (7500nM) and GTE (55.6uM). We also observed that RR CLL cohort exhibited significantly increased drug resistance to most of the tested drugs in the presence of BMSCs with corresponding increases in IC50s (with vs. without BMSCs, fold-change respectively): LP-118 3.92-fold, venetoclax 1.98-fold, carfilzomib 2.58-fold, TP-0903 1.65-fold, panobinostat 204.23-fold, TCIP1 1.91-fold, crenolanib 1.31-fold, idasanutlin 1.69-fold, belinostat 6.14-fold, LP-168 1.29-fold, eprenetapopt 3.44-fold and GTE 1.08-fold (11 out of 12 with p-values <0.05 except for GTE which was not significant, Mann-Whitney U test). We found that even for three drugs with the strongest killing effect based on IC50 levels (LP-118, venetoclax, carfilzomib), their killing effects were significantly reduced in the presence of BMSC (p=0.0002, 0.0002, and <0.0001, respectively). Conclusions: Our results highlight the spectrum of currently available drugs that show promise for therapeutic use in patients with RR CLL who experience disease progression on BTKi. The killing impact was still seen, albeit diminished, even in the presence of BMSC. Our ongoing work will be to associate the drug profiles with each patient's clinical history, as well as to the comprehensive genetic and epigenetic features of the CLL B cell clone.