TP53 Mutations and Other Cell-Intrinsic Determinants of the Apoptotic Response to Ibrutinib in Chronic Lymphocytic Leukemia
Authors: Amin Nisar A. , Balasubramanian Sriram , Saiya-Cork Kamlai , Malek Sami N. 
Published: October 2019

Abstract
Introduction: Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p (all lines). Overall response rates to ibrutinib in treatment naïve or relapsed CLL are high and most patients experience prolonged remission durations, although cases of acquired ibrutinib resistance have been reported.
Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment cell interactions. Weakening of these interactions underlies some of the mechanisms by which ibrutinib is working in vivo. However, comparatively little is known about CLL cell-intrinsic determinants of apoptotic responses induced by ibrutinib. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance and ultimately clinical outcome are warranted.
Methods: We first characterized 50 paired CLL samples procured before and after standard CLL therapies (predominantly chemo immunotherapies) for IGVH status, ZAP70 positivity, CD38 expression, TP53 mutations and FISH and analyzed the relapsed samples by whole exome sequencing (WES). Preliminary ibrutinib treatment studies using CD40-expressing fibroblasts or IL-4 or both in CLL co-culture resulted in low ibrutinib activity, precluding measurements of cell-intrinsic response determinants. Therefore, to assay ibrutinib-mediated CLL cell kill, CLL samples were purified using negative selection and incubated in RPMI medium supplemented with 10% FCS for 72 hours with ibrutinib at varying doses (0.25 µM - 5 µM). Cell death and viability was measured using AnnexinV-PI staining and flow cytometry. IC50 values were calculated using the program XLfit.
Results: CLL cells demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.37 µM - 9.69 µM in pre-treatment samples and 0.56 µM - >10 µM in post treatment samples (mean IC50 values for pre-treatment and relapsed samples were 2.7 µM and 3.7 µM, respectively). Multiple cell-intrinsic determinants of IC50 values to ibrutinib were identified; CLL samples from IGVH unmutated samples were more sensitive than samples from IGVH mutated samples (mean IC50 values for pre-treatment samples were (IGVH -UM) 2.1 µM and (IGVH -M) 3.5 µM (p=0.01), and relapsed samples of (IGVH -UM) 2.8 µM and (IGVH -M) 4.0 µM (p=0.03), respectively, consistent with a greater dependency/signaling capacity of the BCR in IGVH-UM CLL. Similar findings were derived for ZAP70 status, with CLL samples with <20% ZAP70 positivity by FACS being less sensitivity to ibrutinib than ZAP70 positive samples (mean IC50 values for relapsed CLL samples were 4.1 µM and 2.8 µM, respectively; p=0.03). Trisomy 12 was also associated with sensitivity to ibrutinib (mean IC50 values for trisomy 12 positive rCLL samples were 2.8 µM and 4.1 µM, respectively; p=0.03).
IC50 results for paired samples demonstrated a strong concordance within paired samples (Pearson correlation coefficient 0.66). However, ~10% of CLL samples were substantially more resistant to ibrutinib at relapse when compared with their paired diagnosis samples. Of these 5 samples, three had acquired mutations in TP53 following prior chemotherapy; a hypothesis-generating finding that acquired TP53 mutations reduce ibrutinib sensitivities. An expansion cohort of 7 additional ex vivo ibrutinib-treated TP53-mutated CLL samples confirmed the novel and clinically relevant finding that TP53 mutated CLL cells are substantially less sensitive to ibrutinib than TP53 wild type cells (mean IC50 values of 6.1 µM and 2.7 µM, respectively; p<0.001)
Conclusion: Ibrutinib ex-vivo treatment of 50 paired CLL samples procured before and after standard CLL therapies identified cell-intrinsic determinants of ibrutinib sensitivity, including IGVH unmutated CLL and trisomy 12 and implicates prior chemotherapy-selected mutations in TP53 as an intrinsic cause of reduced response. Expansion data identified TP53 mutations as a cell-intrinsic ibrutinib resistance factor in CLL. This novel finding may explain observed shorter remission durations for CLL patients with del17p/mTP53 undergoing ibrutinib therapy. Ex vivo data overall appear concordant with findings in patients treated with ibrutinib thus externally validating our approach and may allow for further refinements in ibrutinib therapy and response prediction.

Disclosures
Balasubramanian: Pharmacyclics LLC, an AbbVie Company: Equity Ownership; Janssen: Employment, Equity Ownership. Malek:Janssen Pharmaceuticals: Research Funding; Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership.