An In Vitro Model for Acute Myeloid Leukemia Relapse Using the SORE6 Reporter

Abstract

Many patients diagnosed with acute myeloid leukemia (AML) relapse within two years of the initial remission. The biology of AML relapse is incompletely understood, although cancer stem-like (CSL) cells have been hypothesized to be important. To test this hypothesis, we employed SORE6, a reporter designed to detect the transcriptional activity of the embryonic stem cell proteins Oct4 and Sox2, to identify/purify CSL cells in two FLT3-mutated AML cell lines. Both cell lines contained ~10% of SORE6+ cells in the steady state. Compared to SORE6− cells, SORE6+ cells exhibited more characteristics of CSL cells, with significantly higher chemoresistance and rates of spheroid formation. SORE6+ cells had substantially higher expression of Myc and FLT3 proteins, which are drivers of SORE6 activity. Using a mixture of SORE6−/SORE6+ cells that were molecularly barcoded, we generated an in vitro study model for AML relapse. Specifically, after ‘in vitro remission’ induced by Ara-C, both cell lines regenerated after 13 ± 3 days. Barcode analysis revealed that most of the regenerated cells were derived from the original SORE6+ cells. Regenerated cells exhibited more CSL features than did the original SORE6+ cells, even though a proportion of them lost SORE6 activity. In bone marrow samples from a patient cohort, we found that relapsed blasts expressed significantly higher levels of Myc, a surrogate marker of SORE6 activity, compared to pre-treatment blasts. To conclude, using our in vitro model, we have provided evidence that CSL cells contribute to AML relapse.

ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia

“…We recently found that purified SORE6 + cells derived from MOLM−13 and MV4−11, two AML cell lines carrying FLT3-ITD, displayed significantly higher CSL phenotype (such as resistance to Ara-C) compared to SORE6 − cells [ 13 ]. Using SORE6 − /SORE6 + cells derived from MOLM−13, we found that the Ara-C inhibitory concentration at 50% (IC50) for SORE6 + cells was 172.9 nM, which is significantly higher than that of their SORE6 − counterparts (61.0 nM, p < 0.0001) ( Figure 1 a).…”

“…We recently generated an in vitro model with features mimicking AML relapse [ 13 ]. Specifically, an ‘in vitro remission state’, defined by the absence of trypan blue-negative cells induced by the lowest required dose of Ara-C, is followed by regeneration of AML cells (i.e., ‘in vitro relapse’) after a period of time.…”

“…Myc has been implicated as a regulator of cancer stemness, as it has been shown to promote pluripotency [ 9 ]. Additionally, we previously found that Myc is higher expressed in SORE6 + compared to SORE6 − cells, and that Myc can regulate SORE6 activity, as inhibition of Myc by shRNA or pharmacological inhibition decreased green fluorescent protein (GFP) expression in SORE6 + cells [ 13 ]. Given the evidence supporting Myc as a regulator of cancer stemness, and our findings that ULK2 may promote the CSL feature of chemoresistance in SORE6 + cells, we asked if the expression level of ULK2 is regulated by Myc.…”

“…We previously generated an in vitro model for disease relapses [ 13 ]. Cells were plated to a concentration of 150,000 cells/mL and were treated with 500 nM Ara-C for two days, which induced ‘in vitro remission’, defined by the absence of trypan blue-negative cells.…”

“…The utility of the SORE6 reporter as an experimental tool to study CSL cells has also been tested and validated in various solid and hematologic cancers [ 10 , 11 , 12 ]. Using SORE6, we previously detected/purified a small subset of SORE6 + cells in two FLT3-mutated AML cell lines and found that SORE6 + cells have significantly higher CSL phenotype than the bulk SORE6 − cells, including chemoresistance [ 13 ]. In the same study, leveraging this SORE6 − /SORE6 + dichotomy, we developed an in vitro AML relapse model, in which regeneration of AML cells was observed after a time period during which no viable cells could be identified using trypan blue; importantly, the regenerated cells were found to be enriched in SORE6 + cells, a finding that is supportive of the role of CSL cells in AML relapse [ 13 ].…”