Food for thought (and CML survival)

In this issue of Blood, Qiu et al¹ shed light on the metabolic changes that arise in a population of chronic myelogenous leukemia (CML) stem cells in a mouse model that allow them to better adapt to continued tyrosine kinase inhibitor (TKI) treatment through HIF1A activity.

CML is a myeloproliferative disease, characterized by leukocytosis and uncontrolled proliferation of granulocytes. The hallmark of CML is the Philadelphia chromosome, in which a reciprocal translocation of t(9;22) results in the BCR-ABL fusion gene and constitutively active tyrosine kinase activity of the resulting oncoprotein. The incorporation of small-molecule TKIs as the first-line treatment for CML has revolutionized prognosis for patients, elevating median survival from a dismal 3 to 5 years from initial diagnosis to that of the general population.² Even so, cases of TKI monotherapies unable to completely eradicate CML persist; imatinib reports molecular relapse in 30% to 60% of patients, and 25% of patients have TKI intolerance.^3,4 This has been attributed to a population of residual leukemic stem cells within CML that functionally share similarities with normal hematopoietic stem cells (HSCs). They are a self-renewing and quiescent population, exhibiting engraftment potential when serially transplanted, and their inert status is thought to contribute to the inadequate clearance of CML and TKI resistance.⁵ The exact details, however, remain elusive. Lifelong therapy imposed on patients with CML undoubtedly calls for better treatment options. In this study, Qiu et al address the metabolic basis for TKI resistance in CML and leave ample room for further speculation on potential intrinsic and extrinsic pathways of therapeutic intervention.

The authors first identified key metabolic changes that occur upon TKI treatment using the double transgenic tetracycline-inducible CML mouse model under the control of the murine stem cell leukemia gene, SCL-tTA/BCR-ABL. After 2 days of treatment with nilotinib, their bioenergetics analysis consistently pointed toward reduced rates of glycolysis and oxidative phosphorylation (OXPHOS) in CML c-Kit⁺ progenitor cells compared with vehicle-treated controls. This was reversed, however, after 2 weeks of treatment, where metabolites of the glycolytic and glutaminolytic cycles and trichloroacetic acid (TCA) intermediates bounced back in CML progenitor cells to levels similar to controls. The authors concluded that CML cells adapt to continued TKI exposure by restoring energy metabolism. To understand the basis for their observations, cells labeled with [U-¹³C₆]-glucose were tracked for their glycolytic end products. The authors found reduced glycolytic (pyruvate, alanine) and TCA cycle intermediates (α-ketoglutarate, fumarate, malate) in CML progenitor cells after 2 days of nilotinib treatment, whereas cells from mice treated for 2 weeks showed increases in lactate and restored glycolytic flux and TCA cycle activity. [U-¹⁵C₅]-glutamine labeling confirmed increase use of glucose carbons for amino acid synthesis.

Single-cell RNA sequencing of untreated Lin⁻Sca-1⁺c-Kit⁺ BM cells showed clustering of quiescent HSC (qHSC) populations with reduced OXPHOS, glycolysis, and nucleotide metabolism and enhanced quiescence gene signatures. These cell populations were enriched after 2 weeks of TKI treatment together with partial restoration of OXPHOS, MYC, and E2 transcription factor following the initial slight reduction observed after 2 days of treatment. Among the metabolic genes upregulated in qHSCs were classical HIF1A targets. Using pySCENIC, a computational method for gene regulatory network (regulon) reconstruction, the authors found significant increase of HIF1A regulon in qHSCs, among various others involved in metabolic regulation. This prompted the authors to investigate the role of HIF1A in maintaining TKI-persistent cells. Combination therapy of nilotinib with the HIF1A inhibitor, echinomycin significantly reduced CML stem and progenitor cells compared with nilotinib treatment alone. This potency was also confirmed for human CML cells, whereby CML CD34⁺ xenografts in immunodeficient NOD/RAG1^−/−IL2Rγ^−/− mice expressing human IL3, CSF2, and KITLG showed reduced CD34⁺CD38⁻CD90⁺ stem cells upon combined nilotinib and echinomycin treatment, validating a critical role of HIF1A activity in maintaining TKI-persistent CML stem cells.

Here, the authors identify HIF1A activity as a key adaptive mechanism that can be targeted to eliminate TKI-persistent CML stem cells, but targeting HIF1A can essentially mean intervention on many other levels, both intrinsic and extrinsic, given its multifaceted role in various physiological pathways (ie, cell proliferation and survival, angiogenesis, innate and adaptive immune activation). The importance of HIF1A activity on stem cell regulation has been established before by Prost et al, who showed that combination therapy of imatinib with the peroxisome proliferator-activated receptor-γ (PPARγ) agonist glitazone effectively eroded CML stem cells by decreasing STAT5 and its downstream target HIF2A.⁶ Furthermore, HIF1A can be stabilized by hypoxia and oxygen-independent pseudohypoxia, both of which are highly relevant for cancer pathobiology.⁷ Regarding the latter, pseudohypoxia-mediated HIF1A dysregulation alone was found to be both essential and sufficient for the pathology of myelodysplastic syndromes.⁸ Of note, hypoxia can also alter epigenetic networks in tumor cells. CML stem cells appear to present a unique pattern of epigenetic hypermethylation, such as upregulating proliferation and antiapoptotic oncogenes via N⁶-methyladenosine as a way to prevent TKI-mediated killing.⁹ In addition, the possible contribution of the bone marrow (BM) microenvironment toward CML persistence should be noted. The authors show upregulation of fatty acid and amino acid oxidation in CML cells, which implies the utilization of other fuel sources besides glucose to supplement their energy demands (ie, from the BM). BM adipocytes could be an ample source of fatty acids for CML stem cells to fuel OXPHOS, as previously reported for acute lymphoblastic leukemia.¹⁰ Alternatively, CML stem cells could actively seek hypoxic surroundings such as the BM sinusoidal network to stabilize HIF1A and thereby promote stem cell function (see figure).

Adaptation is an essential program for survival, whether through intrinsic or extrinsic means. Here, CML stem cells “step up” their metabolism to fend off TKI toxicity. In summary, Qiu et al establish a critical link between HIF1A signaling and metabolic status that can be further explored to search for alternative intrinsic and extrinsic avenues for CML stem cell eradication and treatment-free remission.

Conflict-of-interest disclosure: The authors declare no competing financial interests.