Repurposing pacritinib to target <i>MYD88</i> ‐mutated Waldenström macroglobulinaemia

MYD88 mutations (MYD88Mut) are present in several B-cell malignancies, including Waldenström macroglobulinaemia (WM) (95%–97%) and activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) (40%). MYD88Mut triggers the formation of a ‘Myddosome’, which leads to the sequential activation of IRAK4/IRAK1, JAK2/STAT3 and HCK/BTK pro-survival signalling.1-5 The importance of BTK to MYD88Mut pro-survival signalling supported the development of both covalent and non-covalent BTK inhibitors and the approval of the covalent BTK inhibitors ibrutinib and zanubrutinib for the treatment of symptomatic WM.6, 7 Despite the high level of activity of BTK inhibitors in WM, acquired resistance due to the emergence of BTKCys481Ser mutations is common.8 BTKCys481Ser mutations lead to the activation of ERK1/2 MAP kinases, which triggers interleukin (IL)-6 and IL-10 and propagates paracrine-mediated BTK-inhibitor resistance.9 Pacritinib is a multikinase inhibitor approved in the United States for the treatment of myelofibrosis. Pacritinib blocks JAK2/STAT3, IRAK1 and c-SRC, a highly homologous kinase to HCK.10, 11 Since pacritinib targets multiple kinases implicated in MYD88Mut pro-survival signalling, we explored the repurposing of this U.S. Food and Drug Administration (FDA)-approved therapeutic for the treatment of MYD88Mut WM. For this study, MYD88Mut WM (BCWM.1, MWCL-1, BCWM.2) and ABC DLBCL (TMD-8, HBL1) cell lines, as well as MYD88Wt Burkitt Lymphoma (Ramos) and plasma cell myeloma (RPMI-8226) cell lines, were used in drug evaluations. BTKCys481Ser-expressing BCWM.1 and TMD-8 cell lines, whose development we previously described, were also evaluated.9 Primary CD19+ lymphoplasmacytic lymphoma (LPL) cells were isolated from the bone marrow (BM) of WM patients and genotyped as before.6, 12 Sample use was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board, following patient consent. Ibrutinib, zanubrutinib, pacritinib and venetoclax were obtained from MedChem Express (Monmouth Junction, NJ). KINOMEscan kinase profiling assays were conducted at Eurofins DiscoverX Corporation (San Diego, CA). Enzymatic activity assays for HCK and BTK were performed using Z-Lyte assays (Thermo Fisher Scientific, Waltham, MA) under adenosine triphosphate (ATP) concentrations at K_m. In vitro cellular efficacy was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) as previously described.12, 13 Dose–response curves were generated using GraphPad Prism. Apoptosis was evaluated using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA). Cells (0.2 × 106 per well) were treated with inhibitors overnight in 96-well plates. At least 10 000 events were acquired using a BD LSRFortessa™ Flow Cytometer, and results were analysed using FlowJo software. For WM patient BM LPL cells (0.2 × 106 per well), apoptosis was assessed using Alexa Fluor® 700 anti-human CD19 antibody (Clone: HIB19, BioLegend) in combination with FITC Annexin V Apoptosis Detection Kit I. To conduct signalling studies, cells were treated for 2 h for western blotting and 6 h for Phosflow analysis. For immunoblotting, the following primary antibodies were used: Phospho-HCK (Tyr522) (PA5-37592, Invitrogen), HCK (Clone E1I7F, Cell Signaling), Phospho-BTK (Y223) (Clone EP420Y, Abcam), BTK (Clone D3H5, Cell Signaling), Phospho-ERK1/2 (Thr202/Tyr204) (Clone D13.14.4E, Cell Signaling), ERK1/2 (Clone 137F5, Cell Signaling), caspase-3 (#9662, Cell Signaling Technology), cleaved caspase-3 (Asp175; #9661, Cell Signaling Technology), caspase-9 (#9502, Cell Signaling Technology), caspase-8 (clone 1C12, Cell Signaling Technology) and GAPDH (Clone 0411, Santa Cruz Biotechnology) as a loading control. Phosflow analysis was performed using the following antibodies: BD Phosflow™ BV421 Mouse Anti-BTK (pY223) (Clone: N35-86, BD Biosciences) and Alexa Fluor® 647 Conjugated Phospho-HCK (Y411) (Clone: E5L3D, Cell Signaling, MA). The effects of pacritinib on cell proliferation and apoptosis were evaluated. Pacritinib induced dose-dependent anti-proliferative activity in MYD88Mut WM (BCWM.1, BCWM.2, MWCL-1) and ABC DLBCL (TMD8, HBL-1) cell lines (Figure 1A). Moreover, following 18 h of treatment, MYD88Mut BCWM.1 WM and TMD8 ABC DLBCL cells demonstrated apoptosis at pharmacologically achievable pacritinib concentrations (0.1, 0.5, 1.0 and 2.0 μM), which increased in a dose-dependent manner (Figure 1B). In contrast, pacritinib did not induce anti-proliferative or proapoptotic effects in MYD88Wt Burkitt lymphoma (Ramos) and plasma cell myeloma (RPMI-8226) cell lines (Figure S1). Pacritinib is known to potently abrogate IRAK1 and JAK2, both of which are key pro-survival signalling molecules for mutated MYD88. Pacritinib also blocks c-SRC, a protein with high homology, particularly in the Src homology (SH)2 and SH3 domains, to HCK.10 We therefore performed a kinome-wide analysis of pacritinib (1 μM) in the KINOMEscan assay from Eurofins that covers 468 kinases. This analysis affirmed the complete inhibition of JAK2, as well as the near complete inhibition (99.5%) of IRAK1 kinase activity (Table S1). Our kinome-wide analysis also showed that pacritinib attenuated HCK (59%) and BTK (86%) activation (Table S1). To further validate these findings, we conducted a SelectScreen kinase profiling assay, confirming the inhibition of BTK and HCK with half-maximal inhibitory concentration (IC50) values of 0.367 and 0.528 μM respectively (Figure S2A). Moreover, pacritinib blocked the phosphorylation of BTK and HCK in MYD88Mut BCWM.1 and TMD-8 cells at a concentration of 0.5 μM (Figure S2B). Acquired resistance to covalent BTK inhibitors is commonly due to mutations in BTKCys481 that disrupt inhibitor binding.8 BTKCys481 mutations lead to reactivation of ERK1/2 signalling in the presence of a covalent BTK inhibitor that permits elaboration of pro-inflammatory cytokines and propagates resistance to neighbouring BTK wild-type tumour cells in a paracrine manner.9 To explore if pacritinib blocks ERK1/2 reactivation in BTKCys481-mutated cells, we treated covalent BTK inhibitor-resistant BCWM.1 and TMD-8 cells that were engineered to express BTKCys481Ser as before.9, 13 Our findings showed that pacritinib abrogated phospho ERK1/2 (pERK1/2) signalling in BTKCys481Ser expressing BCWM.1 and TMD8 cell lines (Figure 2A). Moreover, following treatment of BTKCys481Ser-expressing BCWM.1 and TMD8 cell lines with pacritinib for 18 h at concentrations of 0.1, 0.5, 1.0 and 2.0 μM, high levels of apoptosis were observed, increasing in a dose-dependent manner (Figure 2B). This effect appeared independent of canonical apoptotic signalling, as pacritinib did not alter the activation of caspase-9, caspase-3 or caspase-8 in either BTKWt-expressing or BTKCys481Ser-expressing BCWM.1 and TMD8 cells (Figure 2C). We next assessed the activity of pacritinib in primary WM cells, alone and in combination with covalent BTK or BCL2 inhibitors for 18 h at 0.5 μM. The patient characteristics for samples used are detailed in Table S2. Seven out of eight patients harboured the MYD88 L265P mutation, and four carried mutated CXCR4. All patients were treatment-naïve. Pacritinib effectively induced apoptosis in primary WM cells, irrespective of MYD88 and CXCR4 mutation status. The combination of pacritinib with venetoclax achieved a significantly higher level of apoptosis over pacritinib alone, which was also driven by the high level of activity of venetoclax (Figure S3). Notably, pacritinib alone did not induce significant apoptosis in CD19− BM mononuclear cells (Figure S3). The findings from this study provide rationale support for the investigation of pacritinib for the treatment of MYD88Mut WM. Pacritinib can impact multiple MYD88Mut pro-survival pathways, including IRAK1, JAK2 and, as suggested by our findings, HCK and BTK appear to also be targeted at pharmacologically achievable levels. Indeed, in our studies, pacritinib showed higher levels of apoptotic activity against primary MYD88Mut WM versus a covalent BTK inhibitor. The broader spectrum of pro-survival signal suppression may have contributed to these findings. Importantly, pacritinib also showed apoptotic activity against MYD88Mut WM and ABC DLBCL cells engineered to express mutated BTKCys481, which suggests the potential to use this agent in patients with acquired covalent BTK-inhibitor resistance. Lastly, pacritinib also showed enhanced tumour cell killing in primary WM cells when combined with the BCL2 inhibitor venetoclax, suggesting the potential for a combined therapeutic strategy. Given that pacritinib has undergone extensive clinical evaluation and was FDA-approved for the treatment of myelofibrosis, repurposing this agent for the treatment of MYD88Mut lymphomas seems promising. Based on these findings, a Phase II study to evaluate pacritinib in relapsed or refractory WM is being initiated by our group (NCT06986174). SPT, SL, SRS and JJC designed the study. AG, KM, JJC and SRS provided clinical samples. SL, AK, XL, JMH, HS, NT, MLG, DP and ALP performed experiments. SPT, SL, ZRH, CJP, JMH and SRS analysed the data. SL and SPT drafted the initial manuscript. All the authors critically reviewed the initial manuscript and approved the final version. Portions of this research were presented at the 12th International Workshop on Waldenström's Macroglobulinemia in Prague, Czech Republic, in 2024, and at the 66th American Society of Hematology Annual Meeting in San Diego, CA, in 2024. This study was supported by NIH grant 2P50CA100707-16A1 and Leukemia and Lymphoma Society grant 6673-24. JJC received research funds or honoraria from AbbVie, AstraZeneca, BeOne, Cellectar, Johnson & Johnson, Loxo, Mustang Bio, Nurix, Pharmacyclics and Schrodinger. SRS received research/consulting funding from ADC Therapeutics, BeiGene, Sobi and AstraZeneca. SPT received research funding or consulting fees from AbbVie/Pharmacyclics, Janssen, BeOne, Eli Lilly, Bristol Myers Squibb and Ono Pharmaceuticals; and is a named inventor of MYD88 and CXCR4 testing for Waldenström macroglobulinaemia and has assigned all interests to their institution. The remaining authors declare no competing financial interests. Data are available upon request. Figure S1. Pacritinib does not suppress proliferation or induce apoptosis in MYD88Wt lymphoma and myeloma cell lines. Figure S2. Pacritinib attenuates BTK and HCK activation in MYD88Mut WM and ABC DLBCL lymphoma cells. Figure S3. Evaluation of pacritinib alone and in combination in primary WM cells. Table S1. KINOMEscan assay data for pacritinib. Table S2. Genomic characteristics and treatment status for WM patients whose bone marrow samples were used in apoptosis assays with pacritinib. 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