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Acute Myeloid Leukemia Leukemic Initiating Cells: Contribution of hypoxia/HIF pathway to chemoresistance and relapse

Periodic Reporting for period 1 - HifLICs (Acute Myeloid Leukemia Leukemic Initiating Cells: Contribution of hypoxia/HIF pathway to chemoresistance and relapse)

Reporting period: 2019-01-01 to 2020-12-31

Acute Myeloid Leukemia (AML) is a heterogeneous disease that originates from diverse genetic alterations. It can be classified into different cytogenetic groups with variable prognosis. Unfortunately, treatment of AML has not improved over the last 30 years and cure rates remain low. Experimental data supports the view that relapse is driven by a specific subpopulation of cells, termed leukemia initiating cells (LICs), which are able to escape cytotoxic therapy and ultimately reboot the disease. LICs have unique properties, some of them shared with normal hematopoietic stem cells (HSCs), such as relative quiescence, resistance to apoptosis and increased drug efflux, which makes them more resilient to chemotherapy. The discovery of molecular pathways that specifically support LICs, while challenging, would represent an important advancement in AML biology.

With the present project, we study the mechanisms leading to LIC quiescence and to exploit them to sensitize LICs to chemotherapy, in the hope of reducing current relapse rates. Quiescence is partially associated with low oxygen levels (hypoxia) in the bone marrow niche where HSCs/LICs reside. Hypoxia maintains cells in a dormant state as a defense mechanism to prevent their exhaustion. In response to hypoxia, cells activate a specific pathway mediated by Hypoxia Inducible Factors (HIFs), which promote the transcription of many hypoxia-regulated target genes and play a key role in regulating numerous biological processes such as cell proliferation, survival and metabolism.

Although the study of HIF inhibition is at its earliest stage, the growing interest for the function of HIF factors in hematological malignancies suggests its important translational value. Therefore, a deeper understanding of the function of HIFs in cells of hematopoietic origin will create a more precise mapping of the complexity of hypoxia signaling in different physiological and pathological conditions.

The data obtained during this project was focused to couple the hypoxia transcriptional signature of LICs with their in vivo function in the most prevalent human AML cytogenetic subgroups in order to assess: i) whether the hypoxia/HIF pathway represents a mechanism for LICs to evade chemotherapy and, ii) LIC heterogeneity in the hypoxic niche. Targeting this pathway in LICs would open new avenues in AML treatment, leading to clinical trials testing HIF-targeting compounds.
Within this project we have evaluated the hypoxia signaling activation of AML leukemic cells through the transcriptional analysis of i) bulk AML cells, using publicly available datasets and ii) an enriched population in LICs using the state-of-the-art technique of the single cell RNA sequencing. We have focused our study in 3 specific cytogenetic AML subgroups (inv(16), t(8;21) and rearranged MLL) to rule out the huge heterogeneity in the diagnosed AML leukemias.
We have performed a deep bioinformatic analysis with data from 19 different patient samples, paying special attention to the metabolism pathways, differentially expressed genes, cell cycle status, differentiation stage, and of course, the hypoxia signaling pathway, in a subpopulation of cells identified as LICs. We have performed this analysis in patient-paired samples collected at the moment of diagnosis and relapse, which have allowed us the study of these cells in the evolution of the disease.
All generated data are a highly valuable material from which we have extracted information of the status of the most undifferentiated cells in the human leukemia, which have opened new questions and avenues to be explored with the goal of improving our knowledge in the AML biology.
Coupled to this transcriptional study, we have performed functional studies in vitro and in vivo with primary AML cells and patient derived xenografts (PDX) cells analyzing the effect of the chemical inhibition of HIF function in combination with cytarabine treatment, the standard of care used for AML. These experiments suggest a sensitization of the AML cells to the cytarabine treatment when combining with the inhibition of the HIF pathway.
How cancer stem cells trigger quiescence has been widely studied in solid tumors. Some efforts have nevertheless been made to understand the role of HIFs in the pathobiology of blood cancers, revealing that its induction favors progression of the disease and modulates overall sensitivity of leukemic blasts. However, studies have been carried out mainly in different mouse models and much less with human cells. While inhibition of HIFs with different drugs has been shown to significantly reduce tumor initiating activity in preclinical studies with leukemia cells, recent published works have shown that this is not always the case, and the effect can depend on the driver mutation.
HIF transcriptional factors display critical leukemia promoting functions and their inhibition may provide an interesting therapeutic option. At the same time, it is possible that HIFs display specific functions in different AML subtypes, as partly suggested by specific functional interactions with fusion proteins. This complexity of HIF signaling in specific leukemia subgroups is a critical aspect that remains to be addressed. While so far most studies have analyzed cell responses upon HIFs inhibition, few data are available on the set of target genes that are regulated by HIFs in different leukemias. It is critical to define the full spectrum of target genes and possible downstream effects that may result from HIF inhibition. Another important aspect that deserves to be investigated is the role of HIF inhibitors as chemosensitizers. Because various reports have indicated that HIF factors promote LICs maintenance and chemoresistance in leukemia, this project suggests that treatment protocols where HIF inhibitors are administered at low doses may suffice to promote activation and proliferation of LICs, thus increasing their sensitivity to cytotoxic therapeutic agents. Finally, as expression studies are beginning to identify leukemia patients that may have higher expression of HIF factors, we suggest that future investigations onto the efficacy of HIF inhibition also evaluate specific sensitivities based on HIFs expression levels. These studies may allow us to identify patients’ categories that increasingly depend on HIFs activity and may especially benefit from the use of these agents.
In this project, we have studied for the first time the interplay and/or independence of a specific genetic alteration and a transcriptomic signature focused on hypoxia-regulated genes in human AML at the single-cell level, and its correlation with relapse after chemotherapy. These results have identified specific sets of activated genes in the resistant LIC population that may lead to new prognostic factors and to the development of new personalized therapies.
Stem cell biology, developmental leukemia and immunotherapy Group