Deciphering the Biological Basis of Acute Myeloid Leukemia Relapse Using a Novel In-Vitro Study Model

• Author / Creator

  Lai, Justine M.Y.

• Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a poor clinical outcome that is largely attributed to disease relapses. Treatment options for relapsed AML patients are limited; after relapse, the median survival is only ~6 months. The biology underlying relapse is not well understood, and mechanistic studies of AML relapse are scarce, largely because appropriate study models are lacking. Nonetheless, there are several interrelated hypothetical models linked to the concept of cancer stem cells (CSCs). CSCs, which represent a very small subset of the leukemic cell population, survive the induction/consolidation chemotherapy, and they emerge and re-populate the entire leukemic population (i.e. relapse) after a period of dormancy (i.e. remission). This concept is difficult to prove as the definition of CSCs remains to be elusive and study models are lacking.

  The key aim of this study was to examine the validity of the CSC model to explain AML relapse. To identify cancer stem-like (CSL) cells, the SORE6 reporter was employed, which has shown to be useful in detecting/purifying CSL cells; however, this study is the first to use SORE6 to investigate AML. In two FLT3-mutated AML cell lines, MOLM-13 and MV4-11, the SORE6-/SORE6+ dichotomy existed, with SORE6+ cells being significantly more CSL than SORE6- cells. These two subsets were employed to develop an in-vitro model mimicking clinical features of AML relapse. Specifically, after the Ara-C induction of ‘zero viability’ in which viable cells were undetectable by direct microscopic examination, cells regenerated on day 18±2 of the experiment. These phenomena mimic clinical remission and relapse and are thus labeled in-vitro remission and in-vitro relapse, respectively. By molecularly barcoding SORE6- and SORE6+ cells, the relative contributions of original SORE6- and SORE6+ cells could be determined at in-vitro relapse, even if they lose/gain SORE6 activity. Barcode analysis revealed that most in-vitro relapse cells were derived from the original SORE6+ cells and exhibited higher CSL features compared to original SORE6+ cells, even though a proportion of them lost SORE6 activity. Using Myc as the surrogate marker for SORE6+ cells, relapsed patient samples showed an expansion of CSL cells.

  Next, different therapeutic agents were tested in the in-vitro relapse model to determine if SORE6+ cells are important to all relapses. 5-azacitidine (AZA) or the combination of AZA and Venetoclax (Ven) (i.e. AZA+Ven) were used since these two regimens were recently tested in a large clinical trial, with AZA+Ven being significantly superior to AZA in prolonging disease-free survival. Correlating with the clinical data, in-vitro relapse occurred on day 13±1 with AZA, whereas in-vitro relapse did not occur at the endpoint (day 30) with AZA+Ven. In contrast to Ara-C, molecular barcoding and flow cytometry analysis of in-vitro relapse cells after AZA and AZA+Ven treatment did not show the expansion of SORE6+ cells. Using metabolomic analysis, ornithine decarboxylase (ODC) was identified as a marker for CSL expansion. ODC was upregulated in the Ara-C condition, but not in the AZA and AZA+Ven conditions, correlating with barcoding analysis. Additionally, ODC expression was heterogeneous in a cohort of 12 relapsed AML patient samples; however relapsed samples had a significantly higher level of ODC compared to initial diagnostic specimens.

  The utility of the in-vitro study model was further examined by investigating the mechanisms underlying the high chemoresistance in SORE6+ cells. The autophagic capacity of SORE6+ cells was examined, as autophagy has been shown to confer chemoresistance. Using standard autophagy assays, SORE6+ cells carried significantly higher autophagic flux after Ara-C treatment, and inhibition of autophagy significantly sensitized SORE6+ cells to Ara-C. Using an autophagy PCR array, ULK2 was identified as an important mediator for the high autophagy capacity in SORE6+ cells. Pharmacologic inhibition of ULK2 significantly sensitized SORE6+, but not SORE6-, cells to Ara-C. Using the in-vitro model for relapse, higher ULK2 expression was found in regenerated AML cells compared to untreated cells, and pharmacological inhibition of ULK2 prevented in-vitro relapse. Finally, ULK2 expression was higher in relapsed compared to initial diagnostic AML specimens.

  In conclusion, while CSL cells appear to be the major contributors to AML relapse in certain settings, CSL cells may not be uniformly important. While further validation studies are required, the generated in-vitro relapse model is useful in developing concepts and hypothetical models to study AML relapse.

• Subjects / Keywords

  • Acute myeloid leukemia
  • Relapse
  • Cancer stemness

• Graduation date

  Spring 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-cap4-r845

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.