Page 154 - Haematologica April 2020

L. Boutin et al.
Acute myeloid leukemia Side Population blasts exhibit a detoxification transcriptional signature
As SP blasts were more chemoresistant in vitro and in vivo than their MP counterparts, we analyzed their tran- scriptomic profiles to highlight a specific signature of these cells. To do this, we sorted SP versus MP blasts from two AML patients after a 3-day co-culture on HD MSC. A global transcriptomic analysis of sorted SP versus MP AML blasts demonstrated an enrichment of genes implicated in a detoxification program in SP cells. Indeed, using the KEEG database, GSEA analysis showed that the expres- sion of genes involved in xenobiotic and drug metabolism via cytochrome P450 or by other enzymes was up-regulat- ed in SP blasts (Figure 6A). Surprisingly, the expression of ABC transporter transcripts was not homogeneously dif- ferentially modulated in SP versus MP cells (Online Supplementary Figure S5), suggesting that, in our experi- mental conditions, SP phenotype induction is not associ- ated with an increase in ABC transporter mRNA level at steady state, as already suggested in other studies,17 but mainly after drug exposure.18,19 We also observed that, oxidative phosphorylation, fatty acid metabolism and ROS pathway signatures of the Hallmark database were enriched in MP blasts (Figure 6B). Surprisingly, while SP phenotype is a common feature of stem cells,12 and the CD34+ CD38– SP blast population is enriched in cells with a CD123+ stem phenotype (Online Supplementary Figure S6) as compared to CD34+ CD38– MP cells, the stem cell tran- scriptional signatures proposed by Eppert et al.20 and Ng et al.21 were preferentially observed in the AML MP popula- tion (Figure 6C).
Discussion
Chemoresistance of leukemia cells is one of the major challenges which hematologists have to face in order to cure AML. Therefore, a better understanding of this process constitutes the keystone of next generation thera- pies. It is now well known that the BM microenvironment is a key actor in leukemia development and resistance to treatment, but the underlying mechanisms are still unclear.
Our present study shows that MSC that play a central role in BM stromal niches sustain the survival and chemoresistance of AML blasts through the induction of an SP phenotype. This chemoresistance mechanism involves activation of ABC transporters, responsible for drug efflux, in a small proportion of leukemia cells after co-culture with MSC. Whereas these transporters were not active or were poorly active in circulating blasts, inter- actions with stromal cells constantly increased the propor- tion of SP cells in the blast population. Interestingly, the ABC transporter activation pattern appears to be patient dependent. While MSC-induced ABC transporter activa- tion is common to all types of AML, we did not found any correlation between specific probe efflux and patient char- acteristics. However, because AML is an heterogeneous group of diseases, a larger patient study cohort is required in order to identify a potential correlation between ABC transporter activation patterns and AML types. Besides a limited expansion capacity and clonogenicity, the capacity of AML MSC to promote SP phenotype on blasts was similar and even better than that of HD MSC. This obser- vation suggests that the BM stroma of patients may be
adapted to promote blast protection/survival and supports the hypothesis of a specific crosstalk between pathologi- cal blasts and MSC.
Previous data from our group have demonstrated that BM MSC modulate the SP phenotype of HSC from HD.13 In the present study, we show that they also control the induction of the SP functionality on AML blasts. Interestingly, SP leukemic blasts are mostly found in the MSC-adherent fraction, suggesting that close contacts between blasts and stromal cells are required for SP pheno- type induction. As for HD samples, this process is partially dependent on α4 integrin. However, in contrast to HD, for half of the patients studied, this induction is not depend- ent, or is so at a lower level, on β1 integrin or CD44, two cell-surface glycoproteins known to be involved in BM niche HSC nesting. BM niches are reported to be crucial for HSC long-term maintenance22–24 and SP cell quiescence.25 In patients, we show that the proportion of quiescent blasts is higher in the stroma induced-SP cell population than in their MP counterparts. Quiescence is often related to chemoresistance, as only proliferating cells are sensitive to anthracyclines or nucleoside analogs. In the current study, we show that SP blasts are more chemoresistant than MP cells, either in vitro or in vivo in PDX models. This result is confirmed by our data showing that sorted SP blasts express a specific transcriptomic profile focused on genes involved in drug and environment stress detoxification,16 known to participate in the chemoresistance process.26 Therefore, SP cell detection could allow chemoresistant blasts to be monitored during the course of the disease and, targeting stroma-blast interactions would reinitiate cell cycling and, consequently, blast sensitization to chemotherapy. As VLA4 and CD44 play a role in the stro- ma-induced SP phenotype in HD HSC, but only partially in AML blasts, it would be interesting to identify specific interactions between MSC and blasts to specifically target leukemia cells. Deep analyses using transcriptomic and interactomic approaches27 focused on MSC, SP and MP cells from AML patients and HD should highlight new pairs of exclusive interactors involved in stroma-blast inter- actions that would be promising drugable targets.
An important clinical concern is the quest for leukemia stem/initiating cells.28 SP cells exhibit some features of stem cells since they are mostly in G012,14,25 and since their ROS level is lower than that of their MP counterparts.29 In our study, the transcriptomic analysis of SP AML blasts does not correlate with the 114 and 17 gene stemness sig- natures reported by Eppert et al. and Ng et al., respectively,20,21 suggesting that SP AML cells are not enriched in such stem cells. Interestingly, Farge et al.7 and Boyd et al.30 demonstrated that chemotherapy-resistant human AML cells are not necessarily enriched in LSC, but exhibit increased fatty-acid oxidation, high oxidative phosphorylation (OXPHOS) gene signature, and up-regu- lated CD36 expression. By associating a reduction in tran- scripts for oxidative phosphorylation, fatty acid metabo- lism and ROS in SP as compared to MP blasts, our results are in line with those from Ye et al., suggesting that chemoresistance is not necessarily restricted to the LSC compartment but rather could be associated to metabolic adaptations induced by their microenvironment.8
By promoting a better survival of blasts, the stroma- induced SP functionality we identified in blasts could rep- resent a new mechanism of CAM-DR. Whereas BM MSC from HD or AML patients exhibit similar survival and SP-
996
haematologica | 2020; 105(4)