Metabolic alterations mediated by STAT3 promotes drug persistence in CML

Leukemic stem cells (LSCs) can acquire non-mutational resistance following drug treatment leading to therapeutic failure and relapse. However, oncogene-independent mechanisms of drug persistence in LSCs are incompletely understood, which is the primary focus of this study. We integrated proteomics, transcriptomics, and metabolomics to determine the contribution of STAT3 in promoting metabolic changes in tyrosine kinase inhibitor (TKI) persistent chronic myeloid leukemia (CML) cells. Proteomic and transcriptional differences in TKI persistent CML cells revealed BCR-ABL-independent STAT3 activation in these cells. While knockout of STAT3 inhibited the CML cells from developing drug-persistence, inhibition of STAT3 using a small molecule inhibitor sensitized the persistent CML cells to TKI treatment. Interestingly, given the role of phosphorylated STAT3 as a transcription factor, it localized uniquely to genes regulating metabolic pathways in the TKI-persistent CML stem and progenitor cells. Subsequently, we observed that STAT3 dysregulated mitochondrial metabolism forcing the TKI-persistent CML cells to depend on glycolysis, unlike TKI-sensitive CML cells, which are more reliant on oxidative phosphorylation. Finally, targeting pyruvate kinase M2, a rate-limiting glycolytic enzyme, specifically eradicated the TKI-persistent CML cells. By exploring the role of STAT3 in altering metabolism, we provide critical insight into identifying potential therapeutic targets for eliminating TKI-persistent LSCs.

2 used as a viability marker. LSR II with BD Diva Software was used to acquire data while FlowJo 10.4.2 (Tree Star) was used for analysis. For Cell Sorting, the whole bone marrow was depleted for Lineage marker positive cells using biotin and streptavidin beads on the autoMACS sorter.
The lineage negative fraction was then stained(3); stem and progenitor cells were sorted using a FACSAria II (BD Biosciences).
Limiting Dilution Assay: Total bone marrow cells were sorted as CD45.2 + from leukemic or treated mice 4 weeks following TKI and/or LLL12. Numbers of cells to be injected per mouse were sorted into individual wells of a 96-well plate containing 10 4 wild-type CD45.1 + bone marrow (rescue cells). The contents of individual wells were injected into lethally irradiated CD45.1 + recipients (600 rads, twice with a 3-hr interval). Peripheral blood was obtained from mice each month for at least 4 months for chimerism as previously described (4,5).
Apoptosis Assay: Annexin V (ThermoFisher) with DAPI as a counter stain was used for the apoptosis assay. Cells were treated with the respective drugs alone or in combination for 6hrs, 24hrs, 3 days or 7 days. For 7 days, the media was changed on day 3. The cells were harvested and the standard protocol was followed for Annexin V assay. The samples were analyzed using flow cytometry.
Cell Cycle and Proliferation Assay: CML sensitive and resistant K562 cells were treated with their respective drug combinations for 3 days. The cells were then pulsed with 10µM EdU for 4hrs. The cells were then harvested, fixed, stained and analyzed according to the manufacturers protocol for Click-iT EdU flow cytometric assay kit (Invitrogen). For proliferation of murine HSCs and LSCs, BrdU was administered intraperitoneally (1mg/mouse), bone marrow cells were obtained 16 hr post-injection, and BrdU incorporation into stem and progenitor populations was analyzed as described before (6).
Colony Forming Assay: Lin -cKit + Sca1 + were sorted from CML and IM treated CML mice and resuspended in MethoCult GF M3434 (Stem Cell Technology) or human CD34 + cells or K562s were resuspended in MethoCult H4435 Enriched (Stem Cell Technology) with imatinib, LLL12 or shikonin, individually or in combination. Colonies were counted and images were taken after 10 days' incubation in hypoxia (human patient and murine) or normoxia (K562).
Western Blot and RPPA: Lysates were prepared as previously described (7). Blots were probed For RPPA, the samples were prepared and analyzed at MD Anderson Cancer Center as previously described (8)(9)(10).
STAT3 CRISPR: STAT3 knock out parental CML cells were prepared by CRISPR/Cas9 gene editing technology as previously described (11). Single-guide RNA targeting STAT3 was created by cloning appropriate primers into PX458 plasmid. sgRNA was designed to target exon 15 of the TEM Imaging: K562 cells were fixed with 2% glutaraldehyde in 0.1M Cacodylate buffer followed by dehydration with 50% ethanol. The cells were then En bloc stained with 1% uranyl acetate in 50% ethanol for 30min in the dark. The stain was rinsed with 50% ethanol and then dehydrated to 100% ethanol thrice. Cells were infiltrated and embedded in Epon 812 resin. Thin sections were cut using a Leica EM-UC6 ultramicrotome and contrasted with uranyl acetate and Reynold's lead citrate. Images were taken using an FEI Tecnai-Spirit electron microscope operating at 80kV and an AMT Biosprint digital camera. Images were analyzed to calculate the mitochondrial area per cell using Image J (1.52a Wayne Rasband, NIH, USA). using PBS. 50mM LLL12 was made using DMSO, diluted to 5mM using PBS and stored in -20.
On the day of treatment, the 5mM stock was diluted to inject 25mg/kg in 100µL of PBS.  The transcriptomic and proteomic data was carried out in triplicates. For scatter plot, two-way ANOVA was used to determine statistical significance with Tukey's multiple comparison. p values < 0.05 were considered statistically significant. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001; ns = not significant. The data is a representative or a pool of mean ± SD from 2-3 independent experiments. Two-way ANOVA was used to determine statistical significance with Tukey's multiple comparison. p values < 0.05 were considered statistically significant. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001; ns = not significant. The data is a representative or a pool of mean ± SD from 2-3 independent experiments. Unpaired student t-test or Two-way ANOVA was used to determine statistical significance with Tukey's multiple comparison. p values < 0.05 were considered statistically significant. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001; ns = not significant. The data is representative or a pool of mean ± SD from 2 independent experiments. Unpaired student t-test or Two-way ANOVA was used to determine statistical significance with Tukey's multiple comparison. p values < 0.05 were considered statistically significant. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001; ns = not significant.