The role of PIM1 in the ibrutinib-resistant ABC subtype of diffuse large B-cell lymphoma

Abstract

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous lymphoma and the most common subtype of non-Hodgkin lymphoma, accounting for roughly 30% of newly diagnosed cases in the United States. DLBCL can be separated into the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, with distinct gene expression profiles, oncogenic aberrations, and clinical outcomes. ABC-DLBCL is characterized by chronically active B-cell receptor (BCR) signaling that can be modulated by Bruton's tyrosine kinase (BTK) activity. Thus, BTK serves as an attractive therapeutic target in this type of B-cell malignancy. Ibrutinib, a first-in-class, orally available covalent BTK inhibitor, has demonstrated clinical activity in several B-cell leukemias and lymphomas. A phase 1/2 clinical trial of single-agent ibrutinib in relapsed and refractory DLBCL patients revealed an overall response rate of 37% in ABC-DLBCL patients. However, responses to kinase-directed therapies are often limited by emerging resistance mechanisms that bypass the therapeutic target. Here we report the discovery of point mutations within the kinase PIM1 that reduce sensitivity to ibrutinib in ABC-DLBCL. These mutations stabilize PIM1 and affect upstream regulators and downstream targets of NF-κB signaling. The introduction of mutant PIM1 into an ABC-DLBCL cell line, TMD8, increased colony formation and decreased sensitivity to ibrutinib. In addition, ibrutinib-resistant cell lines generated by prolonged ibrutinib exposure in vitro upregulated PIM1 expression, consistent with a role for PIM1 in antagonizing ibrutinib activity. The combination of a pan-PIM inhibitor with ibrutinib synergistically inhibited proliferation in vitro and tumor growth in vivo. Together, these data provide a rationale for combining BTK and PIM1 inhibition in the treatment of ABC-DLBCL.

Keywords: Bruton’s tyrosine kinase; DLBCL; PIM1; ibrutinib.

Figure 1

PIM1 mutations identified in patients with poor responses to ibrutinib increased protein stability. A. Mutation sites of PIM1 identified in ABC- and GCB-DLBCL patient samples. B. Representation of the PIM1 protein showing localization of the mutations observed. C. HEK 293T cells were transiently transfected with WT or mutant PIM1 (L2V, P81S, S97N) constructs, treated with cycloheximide (CHX), and harvested at the indicated time points for immunoblot analysis with anti-PIM1 or anti-β-actin antibodies. D. Semi-quantification using β-actin as a loading control and relative PIM1 levels at time 0 hour as 1.0 were used to determine protein stability.

Figure 2

Mutant PIM1-transduced TMD8 cells had increased colony formation and were more resistant to ibrutinib treatment. (A) WT or mutant PIM1-transduced TMD8 cells were plated at 1×10⁵ per well, and cell number was counted at indicated time points. (B) WT or mutant PIM1-transduced TMD8 cells were plated as in (A) and trypan blue exclusion was used to detect cell viability. (C) WT or mutant PIM1-transduced TMD8 cells were plated in 0.9% MethoCult (3,000 cells/well), and colony formation was scored after 7 days. Graphs represent quantifications of 3 wells, expressed as mean ± SD. (D) Photomicrographs of typical colonies generated by WT or mutant PIM1-transduced TMD8 cells. (E) WT or mutant PIM1-transduced TMD8 cells were treated with indicated concentrations of ibrutinib for 3 days, and the drug effect on cell growth was determined by CellTiter-Glo Luminescent Cell Viability Assay. (F) WT or mutant PIM1-transduced TMD8 cells were plated in 0.9% MethoCult (3,000 cells/well) with indicated concentrations of ibrutinib, and colony formation was scored after 7 days. Graphs represent quantifications of 3 wells and normalizations to the vehicle-treated samples.

Figure 3

PIM1 mutations affected gene expression levels of upsteam regulators and downstream targets of the NF-κB pathway. (A-F) TaqMan® Array Human NF-κB Pathway was used to analyze the expression levels of the indicated genes. GAPDH, HPRT1, GUSB, and 18S were used as internal controls. Relative gene expression indicates the ratio of average gene expression in PIM1-L2V, PIM1-P81S, and PIM1-S97N cells to WT PIM1-expressing cells. Gene expression of downstream targets is shown in (A, B), while gene expression of upstream regulators in NF-κB signaling pathways is shown in (C-F). (G, H) GSEA analyses demonstrate that the expression of genes associated with the Toll-like receptor (TLR) signaling pathway (G) and the IL-1 receptor (IL1R) pathway (H) is enriched in mutant PIM1-transduced TMD8 cells compared with WT PIM1-transduced cells. (I-K) Higher gene expression levels of TLR4 (I), IL1R1 (J), and IL1B (K) were detected in the tumors from ABC-DLBCL patients with poorer responses to ibrutinib (SD + PD). A rank-based statistic (RankProd) was used to determine the significance (P<0.001).

Figure 4

Ibrutinib-resistant ABC-DLBCL cells had higher PIM1 expression levels. A. ABC-DLBCL cell lines had higher gene expression levels of PIM1, PIM2, and PIM3. ABC-DLBCL cell lines (TMD8, HBL1, OCI-LY3, OCI-LY10, U2932, and SUDHL2) and GCB-DLBCL cell lines (SUDHL1, SUDHL4, SUDHL5, SUDHL6, SUDHL8, SUDHL10, DB, RL, Toledo, WSU-NHL, RCK8, OCI-LY8, OCI-LY19, and HT) were used in this study. Relative gene expression of PIM1, PIM2, and PIM3 was normalized to GAPDH by quantitative reverse transcription polymerase chain reaction (qRT-PCR). B. To confirm ibrutinib resistance, cells were treated with indicated concentrations of ibrutinib or vehicle for 3 days, and drug effect on cell growth was determined by CellTiter-Glo Luminescent Cell Viability Assay. C. PIM1 was significantly increased in the ibrutinib-resistant TMD8 cells compared with the parental TMD8 cells from microarray analyses. D. Higher gene expression levels of PIM1/2/3 in the ibrutinib-resistant TMD8 cells was confirmed by qRT-PCR. E. Higher PIM1 protein expression was identified in ibrutinib-resistant (ibR) TMD8 and HBL1 cells compared with parental (P) cells.

Figure 5

The combination of ibrutinib and AZD-1208 synergistically suppressed cell growth and reduced both colony formation and tumor growth. (A) Parental HBL1 cells were treated with indicated concentrations of ibrutinib combined with AZD-1208 (1,000 nM) or vehicle for 3 days, and drug effect on cell growth was determined by CellTiter-Glo Luminescent Cell Viability Assay. (B) Drug dose matrix data of parental HBL1 cells. The numbers indicate the percentage of growth inhibition of cells treated with the corresponding compound combination relative to vehicle control-treated cells. The data were visualized over the matrix using a color scale. (C) Isobologram analysis and synergy scores of the data in (B). (D) HBL1 cells were plated in 0.9% MethoCult (1,000 cells/well) with vehicle, ibrutinib (100 or 1,000 nM), AZD-1208 (100 or 1,000 nM), or the combinations, and colony formation was scored after 7 days. Graphs represent quantifications of 3 wells, expressed as mean ± SD. (E) HBL1 tumor cells were implanted into CB17 SCID mice, and the indicated drugs were orally administered daily when the tumors reached 150 mm³. Tumors were measured twice a week. Significant tumor suppression was observed in the group treated with the ibrutinib/AZD-1208 combination compared with the vehicle-treated group (*P<0.01, repeated measures MANOVA-adjusted univariate F-test).

Figure 6

The combination of ibrutinib and AZD-1208 synergistically suppressed cell growth in ibrutinib-resistant and mutant PIM1-expressing cells. (A) Ibrutinib-resistant HBL1 cells were treated with the indicated concentrations of ibrutinib combined with AZD-1208 (1,000 nM) or vehicle for 3 days, and drug effect on cell growth was determined as in (A). (B) Drug dose matrix data of ibrutinib-resistant HBL1 cells. (C) Isobologram analysis and synergy scores of the data are shown in (B). (D) CI of ibrutinib and AZD-1208 combination in parental HBL1 and ibrutinib-resistant HBL1 cells. The CIs of different concentrations of ibrutinib combined with 1,000 nM AZD-1208 are shown. (E) Mutant PIM1-transduced TMD8 cells were treated with the indicated concentrations of ibrutinib combined with AZD-1208 (2 or 8 μM) or vehicle for 3 days, and drug effect on cell growth was determined. (F) CI of ibrutinib and AZD-1208 combination in mutant PIM1-transduced TMD8 cells. The CIs of different concentrations of ibrutinib combined with AZD-1208 at indicated concentrations is shown.

Figure 7

Hypothetical model of ibrutinib in combination with a PIM inhibitor in ABC-DLBCL cells. Upregulation of gene expression and protein stabilization induced by mutations contribute to the increased protein level of PIM1, which further controls upstream regulators of NF-κB, such as TLR4/7 and IL1R1. In the ibrutinib-resistant cells, combined inhibition of BTK and PIM1 may provide better efficacy.