ALDH1 Activity Identifies Chemotherapy-Resistant Multiple Myeloma Stem Cells
Authors: Zhan Fenghuang , Zhou Wen , Zangari Maurizio , Xu Hongwei , Tricot Guido J. 
Published: October 2019

Abstract
Abstract 990

Background:
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.

Materials and Methods:
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-1st and pre-2nd, post-2nd 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.

Results:
ALDH1 expression increases in the samples post-treatment compared to those at baseline using the serial GEPs obtained pre-1st, pre-2nd, and post-2nd 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).

Conclusion:
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.

Disclosures:
No relevant conflicts of interest to declare.