Bone marrow stromal cells induce an ALDH+ stem cell-like phenotype in AML cells through TGF-β-p38-ALDH2 pathway

Mesenchymal stromal cells (MSCs) in the bone marrow (BM) microenvironment have been shown to induce chemotherapy resistance in acute myeloid leukemia (AML) cells, but the mechanism is not clear. We hypothesized that stromal cells induce a stem-like phenotype in AML cells, thereby promoting tumorigenicity and chemotherapy resistance. We found that aldehyde dehydrogenase (ALDH), an enzyme that is highly expressed in hematopoietic as well as leukemic stem cells was dramatically activated in AML cells co-cultured with BM-MSCs mainly through upregulation of a specific isoform, ALDH2. Mechanistic studies revealed that stroma-derived TGF-β1 induced an ALDH+ phenotype in AML cells via the non-canonical TGF-β pathway through p38 activation. Inhibition of ALDH2 using specific inhibitors significantly inhibited BM-MSC-induced ALDH activity and sensitized AML cells to chemotherapy. Collectively, our data indicate that BM stroma induces a stem-like phenotype in AML cells through the non-canonical TGF-β pathway. Inhibition of ALDH2 sensitizes AML cells to chemotherapy. Impact Statement Currently there is no standard therapy for AML. In this study we identified the mechanism of chemotherapy resistance in AML cells and discovered TGF-β-p38-ALDH2 signaling pathway as a therapeutic target.


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The bone marrow microenvironment (BME) contributes to Acute Myeloid Leukemia (AML) 52 growth and chemotherapy resistance (1, 2). Mesenchymal stromal cells (MSCs) in the bone 53 marrow (BM) are critical for growth induction and anti-apoptotic signaling in AML (3). 54 However, the mechanisms of stroma-mediated AML growth and chemotherapy resistance are not 55 clear. As leukemogenesis and chemotherapy resistance are characteristics of AML stem cells, we 56 hypothesized that the BME induces a stem cell-like phenotype in AML cells. 57 Several signaling pathways contribute to chemotherapy resistance in AML through 58 induction of a high-mesenchymal stem-like cell state (4). Among them, transforming growth 59 factor-β (TGF-β)-mediated canonical and non-canonical pathways have been well-characterized 60 in AML cells (5)(6)(7). Inhibition of TGF-β signaling using small molecule inhibitors or receptor-61 blocking antibodies inhibited leukemia growth and sensitized AML cells to chemotherapy (5). 62 TGF-β signaling has cell type-specific effects and has been involved in the induction of a stem 63 cell-like phenotype in solid tumors (8)(9)(10)(11). 64 Aldehyde dehydrogenase (ALDH) is an enzyme involved in oxidizing toxic aldehydes 65 into neutral acids (12). ALDH activity is increased in hematopoietic stem cells and leukemia 66 stem cells (13,14). ALDH-positive (ALDH + ) leukemia cells have higher tumorigenicity and 67 chemotherapy resistance compared to ALDH-negative cells (14,15). Additionally, high ALDH 68 activity at diagnosis predicts relapse in a subset of AML patients (16). Among the 19 ALDH 69 isoforms identified in humans, the most prominent are ALDH1 isoforms (ALDH1A1-3, 70 ALDHB1, and ALDH1L1&2) and the ALDH2 isoform. ALDH1 family isoforms are located in 71 the cytoplasm, whereas ALDH2 is located in the mitochondria (17,18). 72 In this report, we investigated the effect of BM stromal cells on AML cells, signaling 73 pathways activated, and therapeutic targets that contribute to chemotherapy resistance in AML 74 4 cells. We identified specific inhibitors that could be used in combination with standard 75 chemotherapy for treatment of AML patients.  Flow cytometry analysis of AML cells cultured alone or co-cultured with MSCs was performed 94 as described before (19). The cells were incubated with fluorochrome-conjugated antibodies for 95 20 minutes. The antibody conjugates used were anti-CD45 conjugated with APC (Cat# 304038, 96 6 fluoride (PVDF) membrane. The membrane was blocked with 5% milk in PBS-T (0.05% 119 Tween-20 in PBS) to prevent nonspecific binding of antibodies. Primary antibody incubation 120 was performed in PBS-T with 1% milk at 4°C overnight (refer to Supplementary Table 1  Mix (Applied Biosystems ® ) as described before (20). All samples were run in triplicates. The  Table 2).    Immunodeficiency (NOD/SCID) mice. Leukemia engraftment and growth rate assessment was 195 performed at 1 and 2 weeks as previously described (19).  For survival analysis, we used Kaplan-Meier estimator to estimate the survival function and log-203 rank test to evaluate the statistical significance. To compare the difference two independent 204 groups, we used Mann-Whitney U-test or Student's T-test to examine the statistical significance.

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For the comparison with two groups with paired data, paired Student's T-test was used. We 206 performed one way ANOVA with Tukey's HSD post hoc test to test the significance in the 207 comparison with more than 2 groups. A linear model with interaction term was also used to 208 evaluate the significance with more than two factors in the experiment. A P-value less than 0.05 209 was regarded as statistically significant.

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To investigate the effect of BM stromal cells on AML cells, we co-cultured AML cells 219 OCI-AML3 and HL60 with or without BM-MSCs for 3 or 5 days and measured ALDH activity.

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To validate stroma-induced ALDH activity in primary AML cells, we analyzed ALDH 225 activity in peripheral blood and BM samples derived from AML patients and found that the 226 11 percentage of AML cells varied between patients and did not correlate with age, sex, white 227 blood cell count, or blast percentage (Supplementary Table 3). Next, patient-derived primary 228 AML cells were cultured with or without MSCs for 3 days and ALDH activity was measured in 229 AML cells. We found that co-culture with MSCs significantly induced ALDH activity in AML 230 cells in all 8 patient samples ( Fig. 2A, 2B). This indicates that BM-MSCs support AML cell 231 growth in vivo and induce the ALDH + stem cell phenotype in AML cells.  Table 2). We found differential expression 237 of ALDH isoforms in AML cells cultured with or without BM-MSCs. Specifically, ALDH1L2 238 and ALDH2 expression was upregulated 3-to 5-fold in OCI-AML3 cells co-cultured with BM-

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MSCs compared to OCI-AML3 cells cultured alone (Fig. 2C). To determine the prognostic 240 significance of these isoforms, we analyzed ALDH1L2 and ALDH2 expression in the TCGA 241 AML dataset, which revealed that ALDH1L2 and ALDH2 are upregulated in 8% and 12% of 242 AML cases, respectively. However, increased expression of ALDH2, but not ALDH1L2, confers 243 a worse prognosis and lower survival rate, suggesting that ALDH2 is a key factor promoting 244 AML disease progression (Fig. 2D). To investigate the mechanism of stroma-induced ALDH activity in AML cells, we co-cultured 248 OCI-AML3 cells with or without BM-MSCs for 3 days. OCI-AML3 cells were FACS sorted and 249 12 gene expression analysis was performed by RNA sequencing. Analysis of differentially 250 expressed genes by the Ingenuity ® pathway analysis tool revealed activation of a TGF-β1-251 associated gene signature in OCI-AML3 cells co-cultured with BM-MSCs compared to OCI-252 AML3 cells cultured alone (Fig. 3A). To validate this, we performed RT-PCR for genes that are 253 differentially regulated by TGF-β1. Genes that are positively regulated by TGF-β1 were 254 upregulated and genes that are negatively regulated by TGF-β1, were downregulated in OCI-255 AML3 cells co-cultured with MSCs compared to cells cultured alone (Fig. 3 B, 3C). Hence,  It has been well-established that TGF-β1 signaling is involved in AML-BME interactions (5,22).

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To validate that TGF-β1 regulates downstream transcriptional activity in AML cells, we 279 measured mRNA expression of TGF-β1 target genes in OCI-AML3 cells treated with 280 recombinant TGF-β1. We found that TGF-β1 target genes were upregulated in cells treated with 281 recombinant TGF-β1 compared to untreated controls (Fig. 4C). Interestingly, we also found that 282 ALDH2, which was upregulated in AML cells upon co-culture with BM-MSCs, was also 283 upregulated at the mRNA and protein levels in cells treated with recombinant TGF-β1 (Fig 4D,   TGF-β pathway in AML cells, we measured phosphorylation of Smad2 and Smad3 in OCI-296 AML3 cells treated with recombinant TGF-β1 and still could not find any activity for these 2 297 proteins (Fig. 4E). Next, we tested phosphorylation of p38, which is activated by TGF-β through 298 its non-canonical pathway. Interestingly, we found strong activity for phospho-p38 in OCI-299 AML3 cells treated with supernatants from BM-MSCs compared to cells treated with medium 300 alone. We also found increased phosphorylation of ERK in OCI-AML3 cells treated with BM-301 MSC supernatants, suggesting activation of the Raf-MEK-ERK pathway in these cells. We

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ALDH2 overexpression has been associated with several malignancies and diseases, leading to 328 the development of specific inhibitors with potential therapeutic benefits (18,26,27). To validate 329 that stroma-induced ALDH activity is mostly due to the ALDH2 isoform, we cultured OCI-330 AML3 cells and treated them with ALDH2 inhibitors diadzin or CVT-10216. Strikingly, 331 treatment with ALDH2-specific inhibitors significantly inhibited ALDH activity in OCI-AML3 332 dose-dependently. The percentage of ALDH + cells decreased from 29% ± 1% in untreated cells 333 to 4% ± 0.5% in cells treated with 50 µM of diadzin. Similarly, the percentage of ALDH + cells 334 dropped from 16% ± 5% in untreated cells to 4% ± 0.2% in cells treated with 2 µM of CVT-335 10216 (Fig. 6A, 6B). Next, OCI-AML3 cells were treated with diadzin (5 µM) or CVT-10216 (1 336 µM) in the presence or absence of recombinant TGF-β1 (5 ng/mL) for 3 days. As expected, 337 when treated with recombinant TGF-β1, the percentage of ALDH + cells in OCI-AML3 cells 338 increased from 23% ± 3% to 63% ± 2%. However, ALDH + cells decreased from 63% ± 2% to 339 45% ± 2% when treated with diadzin, suggesting that diadzin inhibits ALDH activity even in the 340 presence of TGF-β1. Similarly, we found an ~50% reduction in ALDH activity in OCI-AML3  In the present study, we demonstrate that BM-MSCs contribute to AML progression and chemo-359 resistance by inducing an ALDH + stem-like phenotype in AML cells. Importantly, through our 360 in-depth gene expression analysis of all 19 ALDH isoforms, we identified ALDH2 as the 361 isoform which is differentially expressed and primarily responsible for the increased ALDH showed that TGF-β1 exerts its effect through a non-canonical/p38-dependent signaling pathway. 375 Hence, our work uncovers a clear link between TGF-β1 secreted by MSCs and the acquisition of 376 a stem-like phenotype of AML cells through ALDH2 overexpression; it also establishes the 377 specific signaling mechanism involved in this interaction.

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ALDH plays a prominent role in several diseases and malignancies (18,29), and specific 379 ALDH inhibitors with potentially anti-tumor effects have been developed (17,26,(30)(31)(32). In this 380 study, we examined the role of ALDH2 inhibitors in counteracting the stem-like phenotype and Our study has some potential limitations. We show promising results regarding ALDH2 388 inhibition as a strategy to counteract AML chemotherapy resistance; however, we have not 389 validated these findings in vivo. Additionally, ALDH2 inhibitors tested significantly reduced, but 390 did not completely inhibit, ALDH activity in AML cells. It remains unclear whether other ALDH 391 isoforms contribute to ALDH activity or the ALDH2 inhibitors we used are not sufficiently      increasing concentrations of ALDH2 inhibitor diadzin. ALDH activity was measured by ALDEFLUOR ® assay using flow cytometry. OCI-AML3 cells were co-cultured with MSCs (200 000 cells/well density) and treated with diadzin (5 µM) for 3 days in the presence or absence of recombinant TGF-β1 (5 ng/mL). ALDH activity was measured as described above.