Ailin Guo

Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Ibrutinib, a Brutons tyrosine kinase inhibitor, is active in CLL, but resistance may emerge. The authors observed the emergence of a CLL clone with a cysteine-to-serine change in amino acid 481 of the target protein that substantially weakens drug binding and leads to resistance.

Characterization of ibrutinib‐sensitive and ‐resistant mantle lymphoma cells

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B‐cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one‐third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib‐sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti‐tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach.

Heightened BTK-dependent cell proliferation in unmutated chronic lymphocytic leukemia confers increased sensitivity to ibrutinib

In chronic lymphocytic leukemia (CLL), patients with unmutated immunoglobulin heavy chain variable region gene (UM-CLL) have worse outcomes than mutated CLL (M-CLL) following chemotherapy or chemoimmunotherapy. However, in the era of BCR-targeted therapies, the adverse prognostic impact of unmutated IGHV seems to be diminishing, and there are clinical datasets showing unexpected improved responses in UM-CLL. We investigated the biological differences of BTK activity between these subgroups and further compared the impact of ibrutinib on molecular and cellular behaviors. Immunoblotting analysis revealed that phosphorylated active BTK is significantly higher in UM-CLL. Moreover, UM-CLL, compared to M-CLL, displayed a much higher proliferative capacity that was correlated with higher phospho-BTK and greater sensitivity to ibrutinib. In addition, BTK depletion with siRNA led to a more prominent reduction in the proliferation of UM-CLL, suggesting that elevated BTK activity is responsible for increased cell proliferation. Further, cell signaling activity by multiple measurements was consistently higher in UM-CLL accompanied by a higher sensitivity to ibrutinib. These studies link UM-CLL to elevated BCR signaling, heightened BTK-dependent cell proliferation and increased sensitivity to ibrutinib. The prognostic significance of IGHV mutation should be reevaluated in the era of new therapies targeting BCR signaling.

Cerdulatinib, a novel dual SYK/JAK kinase inhibitor, has broad anti-tumor activity in both ABC and GCB types of diffuse large B cell lymphoma

B-cell receptor (BCR) and JAK/STAT pathways play critical roles in diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the anti-lymphoma activity of cerdulatinib, a novel compound that dually targets SYK and JAK/STAT pathways. On a tissue microarray of 62 primary DLBCL tumors, 58% expressed either phosphorylated SYK or STAT3 or both. SYK and STAT3 are also phosphorylated in a panel of eleven DLBCL cell lines although ABC and GCB subtypes exhibited different JAK/STAT and BCR signaling profiles. In both ABC and GCB cell lines, cerdulatinib induced apoptosis that was associated with caspase-3 and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest, accompanied by inhibition of RB phosphorylation and down-regulation of cyclin E. Phosphorylation of BCR components and STAT3 was sensitive to cerdulatinib in both ABC and GCB cell lines under stimulated conditions. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in primary GCB and non-GCB DLBCL tumor cells that were accompanied by cell death. Our work provides mechanistic insights into the actions of cerdulatinib, suggesting that the drug has a broad anti-tumor activity in both ABC and GCB DLBCL, at least in part by inhibiting SYK and JAK pathways.

Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL

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 patients 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.

Clonal evolution underlying leukemia progression and Richter transformation in patients with ibrutinib-relapsed CLL

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 BTKC481 , we identified BTKT316A , 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.

Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment

Ibrutinib (BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Notably, cerdulatinib blocked proliferation of ibrutinib-resistant primary CLL cells and of BTKC481S-transfected/ibrutinib-resistant lymphoma cells. These anti-tumor effects are well correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NF?B. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition.

HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells in the hematopoietic system and lymphoid tissues. Although inhibitors targeting the B-cell receptor (BCR) pathway have been successful in the treatment of the disease, the underlying mechanisms leading to BCR over-activity in CLL are not fully understood. In this study, we found that HSP90, a highly conserved molecular chaperone, is overexpressed in CLL compared with resting B cells. HSP90 overexpression is accompanied by the overexpression of several BCR kinases including LYN, spleen tyrosine kinase, Bruton tyrosine kinase and AKT. Chemical and immune-precipitation demonstrated that these BCR constituents are present in a multi-client chaperone complex with HSP90. Inhibition of HSP90 with PU-H71 destabilized the BCR kinases and caused apoptosis of CLL cells through the mitochondrial apoptotic pathway. Further, PU-H71 induced apoptosis in the presence of stromal co-culture or cytoprotective survival signals. Finally, genetic knockdown of HSP90 and its client AKT, but not BTK, reduced CLL viability. Overall, our study suggests that the chaperone function of HSP90 contributes to the over-activity of the BCR signaling in CLL and inhibition of HSP90 has the potential to achieve a multi-targeting effect. Thus, HSP90 inhibition may be explored to prevent or overcome drug resistance to single targeting agents.

Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

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.