HIF-1a Pathway, As a Signal Funnel for Genetic, Epigenetic, and Metabolic Aberrations, Is Sufficient and Essential for MDS Development

Myelodysplastic Syndromes (MDS) are heterogeneous clonal hematopoietic disorders, which are characterized by ineffective hematopoiesis and uni- or multi-lineage dysplasia. Despite the fact that a variety of genetic, epigenetic, and metabolic aberrations have been identified, the clinical features for MDS remain similar, indicating that there is a common underlying mechanism for MDS pathogenesis. Accumulating clinical and research evidence has shown the involvement of systemic inflammation and activated immune signaling in MDS pathogenesis. Hypoxia inducible factor-1α (HIF-1α) is a critical transcription factor for the hypoxic response, angiogenesis, and cancer development. Importantly, HIF-1α is also a key regulator for immune cell activation.

To determine whether MDS patients have an activated HIF-1α signature, we analyzed a public gene expression array data set of CD34⁺ BM cells from a large cohort of MDS patients (n = 183). Although HIF-1α mRNA remains unchanged, some HIF-1α target genes relating with survival/cell growth, glucose metabolism, and invasion/metastasis were significantly activated in the broad spectrum of MDS. We further determined HIF-1α expression in the BM biopsies from MDS patients (n = 39) using immunohistochemistry staining. We found a high frequency of HIF-1α expressed cells in both low- and high-risk IPSS groups. Consistent with this human data, deficiency of frequently mutated genes in MDS resulted in an activated HIF-1α signature in mice. We found that the levels of Hif-1α protein in the c-Kit⁺ BM cells from Dnmt3a-KO, Runx1-KO, and Mll-partial tandem duplication (PTD) knock-in (Mll^PTD/WT) mice were significantly upregulated compared with those from WT controls. Although the expression level of Hif-1α protein in the c-Kit⁺ BM cells from Tet2-KO mice was not changed, they had an activated HIF-1α signature. IDH1/2 mutations are also known to elevate Hif-1α proteins. These results suggest that the HIF-1α pathway is widely activated in MDS patients by MDS-genic mutations through different mechanisms.

To determine whether HIF-1α is sufficient for developing MDS phenotypes, we generated blood specific inducible HIF-1α transgenic mice. Using Vav1-Cre/Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-1α can be induced in a doxycycline dependent manner. HIF-1α-induced mice developed thrombocytopenia, leukocytopenia, and macrocytic anemia. We found micromegakaryocyte and multi-segmented megakaryocyte formations which are a major diagnostic criteria for MDS. In the BM of anemic mice, we found significant reduction of basophilic and polychromatic erythroblasts. The central macrophages are known to form erythroblastic islands that play a critical role in the late stage of erythropoiesis. Both the frequency and absolute number of F4/80⁺ Ter119⁺ BM erythroblastic islands were significantly decreased in HIF-1α-induced mice compared to the control mice. We also found activation of both innate and adaptive immunity in HIF-1α-induced mice. Lineage specific HIF-1α induction revealed the cell-intrinsic effect of HIF-1α on aberrant megakaryocytopoiesis and cell-extrinsic effect of HIF-1α on macrocytic anemia development.

We have previously shown that Mll^PTD/WT mice develop several MDS-associated features, such as increased self-renewal and apoptosis in HSPCs, expansion of myeloid progenitors, and skewed myeloid differentiation. Synergistic effects between MLL-PTD and RUNX1 mutant (S291fs) or Runx1 deletion, further accumulated the Hif-1α protein and caused a variety of MDS features that phenocopy the human MDS. To understand the role of HIF-1α in the context of MDS pathogenesis, we took genetic and pharmacologic approaches. Hif-1α deletion significantly abrogated disease development and rescued macrocytic anemia and thrombocytopenia. This is not due to the elimination of donor derived cells. After the deletion of Hif-1α, a majority of donor derived cells in PB were still CD11b⁺ cells. HIF-1α inhibition significantly reduced colony formation of Mll^PTD/WT/RUNX1-291fs cells and Mll^PTD/WT/Runx1^D/D cells in vitro, and prolonged survival of MDS mice in vivo.

In conclusion, we identified the sufficient and essential role of HIF-1α in developing MDS. These findings implicate that HIF-1α pathway may be the common underlying mechanism of MDS pathophysiology and could be an effective therapeutic target for a broad spectrum of MDS patients.