Targeting HIF-Hydroxylases Compromises Disease Initiation and Propagation, and Synergises with Current Therapies to Eliminate Cancer Stem Cells in AML

Acute myeloid leukaemia (AML) is an aggressive disorder of haematopoietic stem and progenitor cells (HSPCs), in which they acquire mutations, resulting in generation of disease fuelling, and ultimately treatment-resistant, leukaemic stem cells (LSCs). Given the hypoxic nature of the bone marrow microenvironment, the role, and pharmacological manipulation of the Hypoxia Inducible Factor (HIF) pathway in normal and malignant haematopoiesis is an area of significant interest. We, and others, previously found that HIF-α, the master regulator of the pathway, is not essential for normal haematopoiesis and its deletion promotes leukaemogenesis. Here we reveal through genetic, pharmacological and omics-based investigations that HIF stabilisation, through inhibition of its regulator PHD2, is a novel potent therapeutic strategy in AML.

First, to genetically dissect to role of PHD2 in the initiation of AML, we transformed Phd2^cKO HSPCs with AML-associated oncogenes and found that AML cells lacking Phd2 were compromised in vitro, established a weaker leukemic burden and initiated AML with a significantly longer latency in vivo. Moreover, employing our iMLL-AF9;shPhd2 mice, which upon DOX activate MLL-AF9 and a short-hairpin targeting Phd2, we found that Phd2 knockdown in vivo significantly delayed the onset of AML. Next, to investigate acute Phd2 depletion in newly diagnosed disease, we transplanted transformed shPhd2 cells, and upon AML detection induced expression of shPhd2. Notably, Phd2 knockdown in established AML cells compromised disease progression and significantly improved survival of leukaemic mice.

Following this, we examined the impact of Phd2 inactivation on HSC function and multilineage haematopoiesis. Phd2^cKO mice displayed no adverse phenotypes, and Phd2-deficient HSCs were able to successfully sustain multilineage haematopoiesis upon transplantation. Moreover, global DOX-inducible Phd2 knockdown had little effect on haematopoiesis. Thus, PHD2 inactivation compromises AML development and maintenance, but does not impact normal haematopoiesis, suggesting an ample therapeutic window for its inhibition.

Given our genetic evidence that PHD2 is a promising target in AML, we explored the potential of its inhibition. We employed a commercially available clinical trial grade PHD inhibitor, as well as our own novel, selective, and potent PHD inhibitor, both of which work through distinct mechanisms. Significantly, both inhibitors compromised the growth and viability of murine AML cells, as well as multiple established human AML cell-lines and primary patient samples with diverse mutational backgrounds. Moreover, we have also discovered that PHD inhibition markedly synergises with AML treatments currently used in the clinic, thus enhancing their anti-leukaemic effect.

Investigating the molecular mechanisms behind our findings, we found HIF-α stabilisation across multiple AML human cell lines following PHD inhibition. Furthermore, we utilised our Hifα^cKO leukaemic cells which, unlike control cells, were not affected by PHD inhibition, indicating that the anti-leukaemic effect of PHD inhibition is indeed HIF-dependent. Next, we performed RNA-seq and proteomic analyses following genetic and pharmacological PHD inhibition. These experiments revealed increased expression of key myeloid differentiation genes, glycolytic pathway, and a consistent upregulation of HIF target genes, including the pro-apoptotic BNIP3. Finally, pharmacological stimulation of apoptosis combined with PHD inhibition displayed a further and profound anti-leukaemic effect.

Taken together, our data strongly indicate that genetic deletion and specific potent pharmacological inhibition of PHD2 can upregulate HIF-dependent pathways, eradicating AML disease in vitro and in vivo, thus unveiling a novel promising therapeutic target.

No relevant conflicts of interest to declare.