Key Points HIF-1α critically regulates the interaction of neoplastic CLL cells with the leukemic microenvironment. HIF-1α is regulated at the transcriptional level in CLL patients and correlates with CXCR4 expression.

Acute promyelocytic leukemia (APL) is epitomized by the chromosomal translocation t(15;17) and the resulting oncogenic fusion protein PML-RARα. Although acting primarily as a transcriptional repressor, PML-RARα can also exert functions of transcriptional co-activation. Here, we find that PML-RARα stimulates transcription driven by HIF factors, which are critical regulators of adaptive responses to hypoxia and stem cell maintenance. Consistently, HIF-related gene signatures are upregulated in leukemic promyelocytes from APL patients compared to normal promyelocytes. Through in vitro and in vivo studies, we find that PML-RARα exploits a number of HIF-1α-regulated pro-leukemogenic functions that include cell migration, bone marrow (BM) neo-angiogenesis and self-renewal of APL blasts. Furthermore, HIF-1α levels increase upon treatment of APL cells with all-trans retinoic acid (ATRA). As a consequence, inhibiting HIF-1α in APL mouse models delays leukemia progression and exquisitely synergizes with ATRA to eliminate leukemia-initiating cells (LICs).

Elucidating the molecular basis of tumor metastasis is pivotal for eradicating cancer-related mortality. Triple-negative breast cancer (TNBC) encompasses a class of aggressive tumors characterized by high rates of recurrence and metastasis, as well as poor overall survival. Here, we find that the promyelocytic leukemia protein PML exerts a prometastatic function in TNBC that can be targeted by arsenic trioxide. We found that, in TNBC patients, constitutive HIF1A activity induces high expression of PML, along with a number of HIF1A target genes that promote metastasis at multiple levels. Intriguingly, PML controls the expression of these genes by binding to their regulatory regions along with HIF1A. This mechanism is specific to TNBC cells and does not occur in other subtypes of breast cancer where PML and prometastatic HIF1A target genes are underexpressed. As a consequence, PML promotes cell migration, invasion, and metastasis in TNBC cell and mouse models. Notably, pharmacological inhibition of PML with arsenic trioxide, a PML-degrading agent used to treat promyelocytic leukemia patients, delays tumor growth, impairs TNBC metastasis, and cooperates with chemotherapy by preventing metastatic dissemination. In conclusion, we report identification of a prometastatic pathway in TNBC and suggest clinical development toward the use of arsenic trioxide for TNBC patients.

Purpose: Retinoic acid-arsenic trioxide (ATRA-ATO) combination therapy is the current standard of care for patients with acute promyelocytic leukemia (APL) carrying the oncogenic fusion protein PML-RARa. Despite the high cure rates obtained with this drug combination, resistance to arsenic is recently emerging. Moreover, patients with APL carrying the PLZF-RARa fusion protein are partially resistant to ATRA treatment. Hypoxia-inducible factor-1a (HIF-1a) activation has been recently reported in APL, and EZN-2208 (PEG-SN38) is a compound with HIF-1a inhibitory function currently tested in clinical trials. This study investigates the effect of EZN-2208 in different preclinical APL models, either alone or in combination with ATRA.Experimental Design: Efficacy of EZN-2208 in APL was measured in vitro by assessing expression of HIF-1a target genes, cell migration, clonogenicity, and differentiation, vis a vis the cytotoxic and cytostatic effects of this compound. In vivo, EZN-2208 was used in mouse models of APL driven by PML-RARa or PLZFRARa, either alone or in combination with ATRA.Results: Treatment of APL cell lines with noncytotoxic doses of EZN-2208 causes dose-dependent downregulation of HIF-1a bona fide target genes and affects cell migration and clonogenicity in methylcellulose. In vivo, EZN-2208 impairs leukemia progression and prolongs mice survival in APL mouse models. More importantly, when used in combination with ATRA, EZN-2208 synergizes in debulking leukemia and eradicating leukemia-initiating cells.Conclusions: Our preclinical data suggest that the combination ATRA-EZN-2208 may be tested to treat patients with APL who develop resistance to ATO or patients carrying the PLZF-RARa fusion protein.

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression.

Hypoxia inducible transcription factors (HIFs) are the main regulators of adaptive responses to hypoxia and are often activated in solid tumors, but their role in leukemia is less clear. In acute myeloid leukemia (AML), in particular, controversial new findings indicate that HIF-1α can act either as an oncogene or a tumor suppressor gene, and this may depend on the stage of leukemia development and/or the AML sub-type.In this study, we find that HIF-1α promotes leukemia progression in the acute monocytic leukemia sub-type of AML through activation of an invasive phenotype. By applying a list of validated HIF-1α-target genes to different AML sub-types, we identified a HIF-1α signature that typifies acute monocytic leukemia when compared with all other AML sub-types. We validated expression of this signature in cell lines and primary cells from AML patients. Interestingly, this signature is enriched for genes that control cell motility at different levels. As a consequence, inhibiting HIF-1α impaired leukemia cell migration, chemotaxis, invasion and transendothelial migration in vitro, and this resulted in impaired bone marrow homing and leukemia progression in vivo. Our data suggest that in acute monocytic leukemia an active HIF-1α-dependent pro-invasive pathway mediates the ability of leukemic cells to migrate and invade extramedullary sites and may be targeted to reduce leukemia dissemination.

The transcription factor HIF-1α is overexpressed in chronic lymphocytic leukaemia (CLL), where it promotes leukaemia progression by favouring the interaction of leukaemic cells with protective tissue microenvironments. Here, we tested the hypothesis that a pharmacological compound previously shown to inhibit HIF-1α may act as a chemosensitizer by interrupting protective microenvironmental interactions and exposing CLL cells to fludarabine-induced cytotoxicity. We found that the camptothecin-11 analogue EZN-2208 sensitizes CLL cells to fludarabine-induced apoptosis in cytoprotective in vitro cultures; in vivo EZN-2208 improves fludarabine responses, especially in early phases of leukaemia expansion, and exerts significant anti-leukaemia activity, thus suggesting that this or similar compounds may be considered as effective CLL therapeutic approaches.

<p>Supplementary Figure 4: Combined effect of EZN-2208 and ATRA on NB4 cell viability and differentiation.</p>