Re-purposing pacritinib to target MYD88 mutated Waldenstrom’s Macroglobulinemia

To the Editor:

MYD88 mutations (MYD88^Mut) are present in several B-cell malignancies, including Waldenström macroglobulinemia (WM) (95–97%) and activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (ABC DLBCL) (40%). MYD88^Mut triggers the formation of a “Myddosome,” which leads to the sequential activation of IRAK4/IRAK1, JAK2/STAT3, and HCK/BTK pro-survival signaling.^1–5 The importance of BTK to MYD88^Mut pro-survival signaling 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 BTK^Cys481Ser mutations is common.⁸ BTK^Cys481Ser mutations lead to the activation of ERK1/2, which triggers IL-6 and IL-10 and propagates paracrine-mediated BTK-inhibitor resistance.⁹

Pacritinib is a multi-kinase inhibitor approved in the United States for the treatment of myelofibrosis. Pacritinib blocks JAK2/STAT3, IRAK1, and SRC, a highly homologous kinase to HCK.^10,11 Since pacritinib targets multiple kinases implicated in MYD88^Mut pro-survival signaling, we explored the re-purposing of this FDA-approved therapeutic for the treatment of MYD88^Mut WM.

For this study, MYD88^Mut WM (BCWM.1, MWCL-1, BCWM.2) and ABC DLBCL (TMD-8, HBL1) cell lines, as well as MYD88^Wt Burkitt’s Lymphoma (Ramos) and plasma cell myeloma (RPMI-8226) cell lines, were used in drug evaluations. BTK^Cys481Ser-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 ATP concentrations at K_m [app].

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 analyzed using FlowJo software. For WM patient bone marrow 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 signaling studies, cells were treated for 2 hours for Western blotting and 6 hours 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 antiproliferative activity in MYD88^Mut WM (BCWM.1, BCWM.2, MWCL-1) and ABC DLBCL (TMD8, HBL-1) cell lines (Figure 1A). Moreover, following 18 hours of treatment, MYD88^Mut 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 antiproliferative or proapoptotic effects in MYD88^Wt Burkitt’s lymphoma (Ramos) and plasma cell myeloma (RPMI-8226) cell lines (Figure S1).

Figure 1. Pacritinib suppresses the proliferation and induces apoptosis of MYD88^Mut WM and ABC DLBCL lymphoma cells.

(A) Dose-response curves of MYD88^Mut cell lines following treatment with pacritinib for 72 hours at the indicated concentrations. Antiproliferative activity was assessed in MYD88^Mut WM (BCWM.1, BCWM.2, MWCL-1) and ABC DLBCL (TMD8, HBL-1) cell lines. (B) Evaluation for apoptosis following 18 hours of treatment of MYD88^Mut BCWM.1 WM and TMD8 ABC DLBCL cells at pharmacologically achievable pacritinib concentrations (0.1, 0.5, 1.0 and 2.0 μM) using Annexin V-FITC/PI staining. Studies were performed at least twice, and representative results are shown.

Pacritinib is known to potently abrogate IRAK1 and JAK2, both of which are key pro-survival signaling molecules for mutated MYD88. Pacritinib also blocks SRC, a protein with high homology, particularly in the SH2 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 (Supplementary Table 1). Our kinome-wide analysis also showed that pacritinib attenuated HCK (59%) and BTK (86%) activation (Supplementary Table 1). To further validate these findings, we conducted a SelectScreen kinase profiling assay, confirming the inhibition of BTK and HCK with IC₅₀ values of 0.367 μM and 0.528 μM, respectively (Figure S2A). Moreover, pacritinib blocked the phosphorylation of BTK and HCK in MYD88^Mut 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 BTK^Cys481 that disrupt inhibitor binding.⁸ BTK^Cys481 mutations lead to reactivation of ERK1/2 signaling in the presence of a covalent BTK-inhibitor that permits elaboration of pro-inflammatory cytokines and propagates resistance to neighboring BTK wild-type tumor cells in a paracrine manner.⁹ To explore if pacritinib blocks ERK1/2 reactivation in BTK^Cys481 mutated cells, we treated covalent BTK inhibitor-resistant BCWM.1 and TMD-8 cells that were engineered to express BTK^Cys481Ser as before.^9,13 Our findings showed that pacritinib abrogated pERK1/2 signaling in BTK^Cys481Ser expressing BCWM.1 and TMD8 cell lines (Figure 2A). Moreover, following treatment of BTK^Cys481Ser-expressing BCWM.1 and TMD8 cell lines with pacritinib for 18 hours 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 signaling, as pacritinib did not alter the activation of caspase-9, caspase-3, or caspase-8 in either BTK^Wt-expressing or BTK^Cys481Ser-expressing BCWM.1 and TMD8 cells (Figure 2C).

Figure 2. Pacritinib blocks ERK1/2 activation and overcomes acquired covalent BTK-inhibitor resistance due to mutated BTK^Cys481.

(A) Western blot analysis for p-BTK, p-HCK and p-ERK1/2 following treatment of wild-type or mutant BTK^Cys481 expressing BCWM.1 or TMD8 cells with DMSO vehicle control, ibrutinib, or pacritinib (0.5 μM). GAPDH served as a loading control. (B) Evaluation for apoptosis following 18 hours of treatment of BTK^Cys481Ser BCWM.1 and TMD8 cells at pharmacologically achievable pacritinib concentrations (0.1, 0.5, 1.0 and 2.0 μM) using Annexin V-FITC/PI staining. Western blot and apoptosis studies were performed at least twice, and representative results are shown. (C) Western blot analysis of full-length and cleaved forms of caspase-3, caspase-9, and caspase-8 following treatment of wild-type or BTK^Cys481-mutant BCWM.1 and TMD8 cells with DMSO, ibrutinib, or pacritinib (0.5 μM).

We next assessed the activity of pacritinib in primary WM cells, alone and in combination with covalent BTK or BCL2 inhibitors for 18 hours at 0.5 μM. The patient characteristics for samples used are detailed in Supplemental Table 2. Seven out of eight patients harbored 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 MYD88^Mut WM. Pacritinib can impact multiple MYD88^Mut pro-survival pathways, including IRAK1, JAK2, and, as suggested by our findings, HCK and BTK appear to be also targeted at pharmacologically achievable levels. Indeed, in our studies, pacritinib showed higher levels of apoptotic activity against primary MYD88^Mut 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 MYD88^Mut WM and ABC DLBCL cells engineered to express mutated BTK^Cys481, which suggests the potential to use this agent in patients with acquired covalent BTK-inhibitor resistance. Lastly, pacritinib also showed enhanced tumor 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 MYD88^Mut 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).

Supplementary Material

Figure S1

Supplementary Figure 1. Pacritinib does not suppress proliferation or induce apoptosis in MYD88^Wt lymphoma and myeloma cell lines. (A) Dose–response curves of MYD88^Mut and MYD88^Wt cell lines following 72-hour treatment with pacritinib at the indicated concentrations. Antiproliferative activity was evaluated in MYD88^Mut ABC DLBCL (TMD8) and MYD88^Wt Burkitt lymphoma (Ramos) and plasma cell myeloma (RPMI-8226) cell lines. (B) Apoptosis assessment following 18-hour treatment of MYD88^Mut ABC DLBCL (TMD8) and MYD88^Wt Burkitt lymphoma (Ramos) and plasma cell myeloma (RPMI-8226) cells with pacritinib at pharmacologically relevant concentrations (0.1, 0.5, 1.0, and 2.0 μM) using Annexin V–FITC/PI staining. All experiments were performed at least twice, and representative results are shown.

Figure S2

Supplementary Figure 2. Pacritinib attenuates BTK and HCK activation in MYD88^Mut WM and ABC DLBCL lymphoma cells. (A) SelectScreen kinase profiling assay at indicated concentrations for HCK and BTK kinase inhibition. Studies demonstrated that pacritinib inhibited HCK with an IC₅₀ of 588 nM and BTK with an IC₅₀ of 367 nM. (B) Phosflow analysis of phosphorylated HCK (p-HCK Y411) and phosphorylated BTK (p-BTK Y223) in MYD88^Mut BCWM.1 and TMD-8 cells treated 6 hours with vehicle control; ibrutinib (0.5 μM) or pacritinib (0.5 μM). Phosflow studies were performed at least twice, and representative results are shown.

Figure S3

Supplementary Figure 3. Evaluation of pacritinib alone and in combination in primary WM cells. Evaluation for apoptosis following 18 hours of treatment of BM derived CD19⁺ WM cells (upper panel) and CD19^– BM mononuclear cells (lower panel) with pacritinib (0.5 μM), zanubrutinib (0.5 μM) or venetoclax (0.5 μM) alone or combinations as indicated. Error bars represent median with range (n = 8 patients). Statistical analysis was performed using two-way ANOVA with Šídák’s multiple comparisons test.

Table S1

Supplementary Table 1. KINOMEscan Assay Data for Pacritinib. Kinome profiling was performed at 1 μM on 468 Kinases.

Table S2

Supplementary Table 2. Genomic characteristics and treatment status for WM patients whose bone marrow samples were used in apoptosis assays with pacritinib.

Data availability:

Data is available upon request.