Tariquidar sensitizes multiple myeloma cells to proteasome inhibitors via reduction of hypoxia-induced P-gp-mediated drug resistance

Abstract

Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

Keywords: Multiple myeloma; P-glycoprotein; drug resistance; hypoxia.

Figure 1. P-gp overexpression positively correlates with HIF-1α expression in MM plasma cells

(A) Gene expression analysis of P-gp (ENSG00000085563) and HIF-1α (ENSG00000100644) in MM patients stratified into HIF-1α^low (n=108), HIF-1α^int (n=430) and HIF-1α^high (n=126) based on the CoMMpass trial dataset. (B) Gene expression analysis of P-gp (ID probe 209993_at) and HIF-1α (ID probe 200989_at) in MM patients stratified into HIF-1α^low (n=98), HIF-1α^int (n=348) and HIF-1α^high (n=113) based on published datasets from the Gene Expression Omnibus by Zhan et al. Results are shown as mean ± standard deviation (s.d.); the statistical significance was assessed by one-way ANOVA (*** p<0.0001).

Figure 2. Hypoxia increases P-gp protein expression and activity in MM cells

(A) Basal expression of P-gp protein across MM cell lines (OPM2, H929, MM.1S and U266) cultured in normoxia (21% O₂; 24 hours) analyzed by flow cytometry and demonstrated as MFI. (B) The expression of P-gp protein in normoxia (21% O₂; black bars) and hypoxia (1% O₂; white bars) for 24 hours analyzed by flow cytometry demonstrated as fold change and normalized to normoxia. P-gp activity shown as intracellular RhoB (C) and Rho123 (D) content in MM cell lines (OPM2, H929, MM.1S and U266) cultured in normoxia and hypoxia for 24 hours measured by flow cytometry and depicted as MFI normalized to unstained cells, relative to normoxic cells. (E) Histograms demonstrating RhoB in normoxia and hypoxia in MM cell lines. Results are shown as mean ± standard deviation (s.d.); the statistical significance was assessed by student t-test and considered significant for values * p<0.05, ** p<0.01 and *** p<0.001.

Figure 3. P-gp upregulation by hypoxia results in resistance to bortezomib and carfilzomib

(A) Cell survival of MM cell lines (OPM2, H929, MM.1S and U266) treated with and without bortezomib (5nM) or (B) carfilzomib (5nM) for 24 hours in normoxic and hypoxic conditions using a MTT assay normalized to untreated (untr) cells. (C) P-gp expression in MM cells treated with bortezomib (5nM) or (D) carfilzomib (5nM) shown as a fold change of P-gp normalized to isotype control and relative to normoxic cells measured by flow cytometry and demonstrated as average from three MM cell lines (H929, MM.1S and U266). Results are shown as mean ± standard deviation (s.d.); the statistical significance was assessed by student t-test and considered significant for values * p<0.05, ** p<0.01 and *** p<0.001.

Figure 4. P-gp inhibition using tariquidar decreases P-gp activity and restores drug sensitivity

(A) Intracellular RhoB content in MM cells treated with tariquidar (5μM) in normoxia and hypoxia for 24 hours measured by flow cytometry and depicted as (A) MFI normalized to unstained cells, relative to normoxic untreated (untr), averaged from 3 cells lines (H929, MM.1S and U266) or (B) histograms demonstrating RhoB efflux in individual cell lines. (C) Cell survival study of MM.1S cells treated with bortezomib (5nM) or (D) carfilzomib (5nM), in the presence or absence of tariquidar (5μM) or their combination, for 24 hours in normoxia and hypoxia using MTT assay normalized to untreated (untr) MM.1S cells. Results are shown as mean ± s.d. and the statistical significance was assessed by student t-test and considered significant for values * p<0.05, ** p<0.01 and *** p<0.001.