Waldenstrom macroglobulinemia cells devoid of BTKC481S or CXCR4WHIM-like mutations acquire resistance to ibrutinib through upregulation of Bcl-2 and AKT resulting in vulnerability towards venetoclax or MK2206 treatment.

Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9-3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTKC481S and CXCR4WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTKC481S and CXCR4WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.

Coinhibition of the deubiquitinating enzymes, USP14 and UCHL5, with VLX1570 is lethal to ibrutinib- or bortezomib-resistant Waldenstrom macroglobulinemia tumor cells.

The survival of Waldenstrom macroglobulinemia (WM) tumor cells hinges on aberrant B-cell receptor (BCR) and MYD88 signaling. WM cells upregulate the proteasome function to sustain the BCR-driven growth while maintaining homeostasis. Clinically, two treatment strategies are used to disrupt these complementary yet mutually exclusive WM survival pathways via ibrutinib (targets BTK/MYD88 node) and bortezomib (targets 20 S proteasome). Despite the success of both agents, WM patients eventually become refractory to treatment, highlighting the adaptive plasticity of WM cells and underscoring the need for development of new therapeutics. Here we provide a comprehensive preclinical report on the anti-WM activity of VLX1570, a novel small-molecule inhibitor of the deubiquitinating enzymes (DUBs), ubiquitin-specific protease 14 (USP14) and ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5). Both DUBs reside in the 19 S proteasome cap and their inhibition by VLX1570 results in rapid and tumor-specific apoptosis in bortezomib- or ibrutinib-resistant WM cells. Notably, treatment of WM cells with VLX1570 downregulated BCR-associated elements BTK, MYD88, NFATC, NF-κB and CXCR4, the latter whose dysregulated function is linked to ibrutinib resistance. VLX1570 administered to WM-xenografted mice resulted in decreased tumor burden and prolonged survival (P=0.0008) compared with vehicle-treated mice. Overall, our report demonstrates significant value in targeting USP14/UCHL5 with VLX1570 in drug-resistant WM and carries a high potential for clinical translation.

Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

Nimbolide targets BCL2 and induces apoptosis in preclinical models of Waldenströms macroglobulinemia.

Neem leaf extract (NLE) has medicinal properties, which have been attributed to its limonoid content. We identified the NLE tetranorterpenoid, nimbolide, as being the key limonoid responsible for the cytotoxicity of NLE in various preclinical models of human B-lymphocyte cancer. Of the models tested, Waldenströms macroglobulinemia (WM) cells were most sensitive to nimbolide, undergoing significant mitochondrial mediated apoptosis. Notably, nimbolide toxicity was also observed in drug-resistant (bortezomib or ibrutinib) WM cells. To identify putative targets of nimbolide, relevant in WM, we used chemoinformatics-based approaches comprised of virtual in silico screening, molecular modeling and target-ligand reverse docking. In silico analysis revealed the antiapoptotic protein BCL2 was the preferential binding partner of nimbolide. The significance of this finding was further tested in vitro in RS4;11 (BCL2-dependent) tumor cells, in which nimbolide induced significantly more apoptosis compared with BCL2 mutated (Jurkat BCL2(Ser70-Ala)) cells. Lastly, intraperitoneal administration of nimbolide in WM tumor xenografted mice, significantly reduced tumor growth and IgM secretion in vivo, while modulating the expression of several proteins as seen on immunohistochemistry. Overall, our data demonstrate that nimbolide is highly active in WM cells, as well as other B-cell cancers, and engages BCL2 to exert its cytotoxic activity.

Separation of peptides by HPLC using a surface-confined ionic liquid stationary phase.

A butylimidazolium bromide surface-confined ionic liquid stationary phase was synthesized in-house. The synthesized phase was investigated for the separation of five peptides (Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin, and angiotensin-II). The peptides were successfully separated in less than 5 min. The effect of trifluoroacetic acid (TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion-pairing agent in separation of peptides on this phase.