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.

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.

<div>Abstract<p><b>Purpose:</b> 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-RARα. Despite the high cure rates obtained with this drug combination, resistance to arsenic is recently emerging. Moreover, patients with APL carrying the PLZF-RARα fusion protein are partially resistant to ATRA treatment. Hypoxia-inducible factor-1α (HIF-1α) activation has been recently reported in APL, and EZN-2208 (PEG-SN38) is a compound with HIF-1α 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.</p><p><b>Experimental Design:</b> Efficacy of EZN-2208 in APL was measured <i>in vitro</i> by assessing expression of HIF-1α target genes, cell migration, clonogenicity, and differentiation, <i>vis a vis</i> the cytotoxic and cytostatic effects of this compound. <i>In vivo</i>, EZN-2208 was used in mouse models of APL driven by PML-RARα or PLZF-RARα, either alone or in combination with ATRA.</p><p><b>Results:</b> Treatment of APL cell lines with noncytotoxic doses of EZN-2208 causes dose-dependent downregulation of HIF-1α <i>bona fide</i> target genes and affects cell migration and clonogenicity in methylcellulose. <i>In vivo</i>, 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.</p><p><b>Conclusions:</b> 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-RARα fusion protein. <i>Clin Cancer Res; 21(16); 3685–94. ©2015 AACR</i>.</p></div>

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

<p>Supplementary Figure 2: High doses EZN-2208 induce apoptosis in leukemic bone marrow cells.</p>

<p>Supplementary Figure 1: EZN-2208 does not impair cell migration and colony formation in U937-MT control cells</p>

<p>Supplementary Figure 3: EZN-2208 affects leukemic stromal cell numbers.</p>