Developing Novel Therapeutic Strategies to Overcome Ibrutinib Resistance in Mantle Cell Lymphoma
Authors: Pham Lan , Zhang Liang , Tao Wenjing , Zhao Donglu , Zhang Hui , Xie Jingmeng , Wang Jack , Nomie Krystle , Wang Michael 
Published: October 2019

Abstract
MCL accounts for 7% of all non-Hodgkin lymphoma cases and is a rare and incurable subtype of B-cell lymphoma. Unfortunately, most MCL patients experience disease progression after frontline therapy, with a median overall survival of approximately 1-2 years after relapse; therefore, novel therapies for MCL are urgently needed. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) was recently approved by the FDA to treat this disease. In our prior multiple-center phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68%, with a median progression free survival (PFS) of 13.9 months, surpassing the effectiveness of other therapies. Based on our phase II clinical trial, approximately 48% of patients showed no response to ibrutinib or displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment. Furthermore, the patients who initially show lengthy, durable responses to ibrutinb often acquire resistance and relapse at a median of 17 months. Once patients relapse after ibrutinib treatment, the 1-year survival rate is only 22%; therefore, the vast majority of MCL patients who experience disease progression after ibrutinib treatment will die within 12 months. Overall, the dismal outcomes of these patients demand that novel and pioneering approaches be taken to overcome ibrutinib resistance and improve the cure rate of MCL. To understand the mechanism(s) of acquired resistance for ibrutinib in MCL cells, we have recently generated two ibrutinib-resistant Jeko-1 and PF-1 MCL cell lines. These resistant cells were generated in culture by growing these cells in increasing concentrations of ibrutinib over a period of six months. The ibrutinib-resistant MCL cell lines were at least 8-10-fold more resistant to ibrutinib in comparison to the parental cell lines. The ibrutinib-resistant MCL cell lines were also cross-resistant to another BTK inhibitor, BGB, suggesting that BTK is the specific target in the resistant cell lines. Whole exome sequencing (WES) did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. Next, we conducted proteomics analyses using reverse-phase protein array (RPPA) to examine the protein expression profile of parental and ibrutinib-resistant MCL cell lines. We discovered a significant increase the levels of PI3K/AKT/mTOR/MCL-1 compensatory pathway components in both Jeko-1 and PF-1 ibrutinib-resistant cell lines when compared with their parental cells. Next, we screened for potential therapeutic agents that can overcome ibrutinib resistance. Of the many key agents tested, we discovered that the proteasome inhibitor carfilzomib (CFZ), the Bcl-2 inhibitor ABT-199, and the mTOR inhibitors BEZ and AZD demonstrated the greatest potential to overcome ibrutinib resistance in MCL cells. Interestingly, the ibrutinib-resistant MCL cells developed sensitization to CFZ. We found that the IC50 values of CFZ in the PF-1 ibrutinib-resistant cells were significantly lower (10-fold) in comparison to the IC50 values in the PF-1 parental line, which is consistent with primary MCL cells, where MCL cells derived from ibrutinib-resistant patients are more sensitive to CFZ than ibrutinib-naïve primary MCL cells. These findings are reflected by the function of the 20S proteasome because ibrutinib-resistant MCL cells have higher 20S proteasome activity. In contrast, MCL cell lines and primary MCL cells are highly sensitive to ABT-199 (IC50 < 50 nM) regardless of ibrutinib sensitivity, and ABT-199 is strictly dependent on Bcl-2 protein expression. Furthermore, based on our RPPA data, BEZ and AZD were used to target mTOR to determine whether mTOR inhibition reverses ibrutinib resistance. Our data showed that ibrutinib-resistant MCL cells are more highly resistant to both BEZ and AZD compared with the parental lines. Taken together, our data indicate that CFZ, ABT-199, and mTOR inhibitors have the potential to overcome ibrutinib resistance, and further investigation with in vivo models is warranted to validate our findings.

Disclosures
Pham: Amgen, Onyx, Millennium: Research Funding. Wang:Celgene: Research Funding.