ALDH1 Activity Identifies Chemotherapy-Resistant Multiple Myeloma Stem Cells

Cancer stem cells are a chemotherapy-resistant subpopulation capable of self-renewal and of regenerating bulk tumor, thereby causing relapse and ultimately patient's death. Aldehyde dehydrogenase 1 (ALDH1) has been found to be a reliable marker for cancer stem cells in many human malignancies. The purpose of this study is to investigate the stem cell–related function and clinical significance of ALDH1 in myeloma.

9 myeloma (MM) cell lines (XG6, KMS12B, ARP1, 8226, U266, H929, JJN3, OPM2, 5T33) were assessed for ALDH1 activity using the ALDEFLUOR® assay. The positive fraction was sorted using FACSAria II. The functional role of ALDH1+ cells in MM was characterized by clonogenic capacity using soft agar assays and tumorigenicity in NOD-RAG/null gamma mice. Gene expression profiling (GEP) comparison between ALDH1+ and ALDH1- cells was performed using the Affymetrix U133Plus2 chips. We also performed GEP on 9 patients including 36 samples at baseline, after chemotherapy (pre-1^st and pre-2^nd, post-2^nd autologous stem cell transplants (ASCT), and pre-consolidation. In addition, a total of 550 samples with GEP and clinical data, obtained from published studies, were analyzed.

ALDH1 expression increases in the samples post-treatment compared to those at baseline using the serial GEPs obtained pre-1^st, pre-2^nd, and post-2^nd ASCT from 9 MM patients. All 9 MM cell lines studied contained a very small subset of cells with ALDH1 activity (ALDH1⁺), among which XG6, KMS12B, ARP1 (human MM cell lines), and 5TGM1 (mouse MM cell line) had the highest fraction of ALDH1⁺ cells (3.63%, 3.67%, 1.7%, and 1.9% respectively). The colony-forming efficiency of ALDH1⁺ cells was almost 2× higher (1390 clones out of 5000 seeding cells) than that of the ALDH1⁻ cells (730 clones out of 5000 seeding cells) in ARP1; p<0.01). The tumorigenicity of FACS-sorted ARP1 ALDH1⁺ and ALDH1⁻ cells was compared by injecting 10,000 cells subcutaneously into the right flank of NOD-Rag/null gamma mice (n=5). After 8 weeks, ALDH1⁺ cells showed a greater tumor forming capacity (4/5 or 80%) than the ALDH1⁻ group (1/5 or 20%) and also a larger average tumor volume (6.80cm³ vs 1.94 cm³). Furthermore, we treated the two groups with the bortezomib for 48 hours and found that ALDH1⁺ cells showed much less sensitivity than the ALDH1⁻ cells (P < 0.01) at different bortezomib concentrations. GEP performed on XG6, KMS12B, and ARP1 human cell lines showed that 20 genes were highly differentially expressed between ALDH1⁺ and ALDH1⁻ fractions. A risk score determination showed that 17 of the 20 genes were up-regulated (UBE2C, CDC2, FAM83D, TOP2A, ASPM, DEPDC1, HJURP, CDCA3, AURKA, TTK, NCAPH, CCNB1, NEK2, KPNA2, KIF14, NDC80 and AURKB) and the remaining 3 were down-regulated (CALU,228697_at, 231597_X_at). Five of the 17 up-regulated genes are linked to drug resistance (NEK2, CDC2, CCNB1 and TOP2A) and the stemness-related Notch pathway (TTK). Kaplan-Meier analyses of event-free and overall survivals were used to determine the correlation of risk score with patient outcome. These revealed inferior outcomes among the 38 patients with a high level of ALDH1 risk score compared to the remaining 313 patients with a low level of ALDH1 risk score in the TT2 trial (p<0.0001) and also among the 31 patients with ALDH1+ compared to the remaining 150 patients with ALDH1⁻ MM cells in the TT3 trial (p=0.0007).

Our data suggest that MM cells contain a minor but more tumorigenic ALDH1⁺ stem cell-like compartment consisting of chemotherapy-resistant cells, and that ALDH positivity by GEP confers inferior survival outcomes in MM patients. Future studies to perform include investigating how ALDH1 activity is linked to the stemness-related Notch pathway and whether inhibiting ALDH1 directly or indirectly is a viable target for novel anti-MM therapy.

No relevant conflicts of interest to declare.