Key Points• Del(18p), together with del(17p)/TP53 mutations, is present at a high frequency before ibrutinib treatment.• BTK mutations drive ibrutinib relapse, but del(17p)/TP53 mutations may be dispensable.Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTK C481 , we identified BTK T316A, a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts.Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

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Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patient's clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

Inhibition of B-cell receptor (BCR) signaling through the BTK inhibitor, ibrutinib, has generated a remarkable response in mantle cell lymphoma (MCL). However, approximately one third of patients do not respond well to the drug, and disease relapse on ibrutinib is nearly universal. Alternative therapeutic strategies aimed to prevent and overcome ibrutinib resistance are needed. We compared and contrasted the effects of selinexor, a selective inhibitor of nuclear export, with ibrutinib in six MCL cell lines that display differential intrinsic sensitivity to ibrutinib. We found that selinexor had a broader antitumor activity in MCL than ibrutinib. MCL cell lines resistant to ibrutinib remained sensitive to selinexor. We showed that selinexor induced apoptosis/cell-cycle arrest and XPO-1 knockdown also retarded cell growth. Furthermore, downregulation of the NFkB gene signature, as opposed to BCR signature, was a common feature that underlies the response of MCL to both selinexor and ibrutinib. Meanwhile, unaltered NFkB was associated with ibrutinib resistance. Mechnistically, selinexor induced nuclear retention of IkB that was accompanied by the reduction of DNA-binding activity of NFkB, suggesting that NFkB is trapped in an inhibitory complex. Coimmunoprecipitation confirmed that p65 of NFkB and IkB were physically associated. In primary MCL tumors, we further demonstrated that the number of cells with IkB nuclear retention was linearly correlated with the degree of apoptosis. Our data highlight the role of NFkB pathway in drug response to ibrutinib and selinexor and show the potential of using selinexor to prevent and overcome intrinsic ibrutinib resistance through NFkB inhibition.