Kinome expression profiling to target new therapeutic avenues in multiple myeloma

Abstract

Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.

Figure 1

Identification of 36 kinome related probe sets linked to prognosis in three independent cohorts of newly diagnosed multiple myeloma patients. (A) Workflow analysis used to identify kinases with gene expression associated with a prognostic value in muliple myeloma (MM). Cohort 1: HM-Montpellier cohort, Cohort 2: UAMS-TT2, Cohort 3: UAMS-TT3. Poor prognosis means that high gene expression is associated with a significant negative outcome, while good prognosis means that high gene expression is linked to a better outcome (B) Reactome molecular signatures significantly enriched in the kinases related to a poor outcome in MM (C) String network of the 36 identified kinases. Red color represents cell cycle related kinases.

Figure 2

Prognostic value of the Kinome Index in multiple myeloma. (A) Clustergram in the 206 HM cohort’s patients (206 patients) of the 36 genes signal used to build the Kinome Index (KI). Signals are displayed from low (deep blue) to high (deep red) expression. (B) Patients of the HM cohort were ranked according to increased KI and a maximum difference in OS was obtained with KI of 2.1 splitting patients into high-risk (31%) and low-risk (69%) groups (OS and EFS). (C) The KI was computed for MMC of patients belonging to the subgroups of the University of Arkansas for Medical Science (UAMS) molecular classification of MM, using UAMS-TT2 cohort. CD1: cyclin D1 and cyclin D3; CD2: cyclin D1 and cyclin D3; HY: hyperdiploid; LB: low bone disease; MF: c-MAF and MAFB; MS: MMSET; MY: myeloid; PR: proliferation; D) KI is increased in Pre-plasmablasts characterized by high proliferation during normal B- to PC-differentiation. MBC: memory B cells (n=5); prePB: pre-plasmablast (n=5); PB: plasmablast (n=5); LLPC: long live plasma cells (n=5); BMPC: bone marrow plasma cells (n=5); HM MM cohort (n=206); TT2 MM cohort (n=345); TT3 MM cohort (n=158); HMCL: human myeloma cell lines (n=44). (E) KI is significantly higher at relapse compared to diagnosis in a cohort of 23 paired patient’s samples (paired T-Test). P-value: *<0.05; **<0.01; ***<0.001.

Figure 3

Selected kinases inhibition induces human myeloma cell toxicity. (A) Selection of seven kinases for biological investigations based on citation report in Pubmed and the availability of inhibitors. (B) IC50 of the different drugs in four human myeloma cell lines (HMCL), and calculated IC20 for the AMO1 HMCL; C) Kinase inhibitors induce apoptosis (annexin V and PARP cleavage) in AMO1 MM cell line at concentrations close to the calculated IC20 and IC50. Annexin, and PARP cleavage, were monitored by flow cytometry after four days of treatments. Results are representative of four independent experiments. Statistical significance was tested using a Student T-Test for pairs. P-value: *<0.05; **<0.01; ***<0.001.

Figure 4

Selected kinases inhibition induces human primary multiple myeloma (MM) cell death and toxicity on 5TMM murine cells. A) Mononuclear cells from five patients with MM were treated or not with CHK1i, MELKi and PLK4i. At day 4 of culture, the viability and total cell counts were assessed and the percentage of CD138+ viable plasma cells and bone marrow non-myeloma cells were determined by flow cytometry. Results are median values of the numbers of myeloma cells in the culture wells. Results were compared with a Student T-Test for pairs. B) Murine myeloma cell (5T33vv) viability was monitored by CTG after 24 and 48 hours treatment with CHK1i, MELKi and PLK4i. Results are representative of three independent experiments C) Apoptosis and Signaling pathways targeted by CHK1i, MELKi and PLK4i. Proteins accumulations were monitored after 48h treatment on AMO1 human myeloma cell lines (HMCL) using proteome profiler array. Relative amount was calculated as the mean of pixel density. P-value: *<0.05; **<0.01; ***<0.001.

Figure 5

Kinase inhibitors enhance the sensitivity of multiple myeloma cells to conventional treatments. Human myeloma cell lines (HMCL) were cultured for four days in 96-well flat-bottom microtiter plates in RPMI 1640 medium, 10% fetal calf serum, 2 ng/mL IL-6 culture medium (control) and graded Melphalan concentrations (A) or Lenalidomide concentrations (B) in presence or absence of IC20 of CHK1i, MELKi, PBKi, CDC7-DBF4i, SRPKi, MPS1i and PLK4i. IC50 were calculated after viability assessment by CellTiter-Glo® Luminescent Cell Viability Assay. Results are representative of three independent experiments. P-value: *<0.05; **<0.01; ***<0.001. S: significant synergy calculated by the method of Chou and Talalay.

Figure 6

Conventional multiple myeloma therapies are potentialized by selected kinase inhibitors. Co-treatment with selected kinase inhibitors at IC20 and Melphalan or Lenalidomide. (A) Apoptosis induction was analyzed using Annexin V APC staining by flow cytometry. (B) DNA damage induction was analyzed measuring ϒH2AX levels; Results are representative of four independent experiments. CI: calculated combination index. Statistical significance was tested using a Student T-Test for pairs. P-value: *<0.05; **<0.01; ***<0.001. ^#Significantly different of each individual treatment.

Figure 7

Kinase inhibitors overcome resistance of Melphalan resistant multiple myeloma cells. (A) Dose response curves of XG7 WT and XG7 MRes cell lines. (B) XG7 WT and XG7 MRes HMCL were cultured for 4 days in 96-well flat-bottom microtiter plates in RPMI 1640 medium, 10% fetal calf serum, 2 ng/mL IL-6 culture medium (control) and graded Melphalan concentrations and selected kinase inhibitors at IC20. At day 4 of culture, the viability was assessed by CellTiter-Glo® Luminescent Cell Viability Assay. Data are mean values ±SD of three independent experiments. P-value: *<0.05; **<0.01; ***<0.001 using a student T-Test for pairs. Mres: Melphalan resistant; SD: standard deviation. WT: wild-type.

Figure 8

Kinome expression profiling to define new therapeutic targets in multiple myeloma. The prognostic impact of the kinome expression was challenged in three independent cohorts of newly-diagnosed multiple myeloma (MM) patients representing 709 patients. 36 clinically relevant genes were selected as potential therapeutic targets, and were used to create a Kinase Index (KI) with a strong prognostic value. Among the 36 selected kinases, we validated seven kinases as new therapeutic targets in MM, as their related inhibitors presented therapeutic interest in MM for personalized treatments.