ALBERT

Description: Abstract 2085 Poster Board II-62 Over the past 10 years, the incidence of acute myeloid leukemia (AML) has increased significantly with approximately 15 000 new cases annually. Standard induction chemotherapy consisting of cytarabine (Ara-C) and an anthracycline induces remission rates between 50% and 85%. Unfortunately, the majority of patients who achieve remission will relapse and die from their disease within 2 years, highlighting the need for novel therapeutic targets. The eukaryotic translation factor (eIF4E) is overexpressed in many human malignancies, including AML, and is associated with poor prognosis as well as clinical progression. Ribavirin, an anti-viral molecule, is classically used in the treatment of hepatitis C (with interferon), SARS, RSV, Lassa fever and influenza. Its structure physically mimics the m(7)G cap of mRNA, thus inhibiting eIF4E-induced export and translation of sensitive transcripts. We are carrying out the first clinical trial targeting eIF4E with ribavirin in AML patients. Clinical and molecular efficacy has been evaluated in 13 patients to date. The treatment was well tolerated by all patients with no marked toxicity observed. Importantly, no patients developed hemolytic anemia. We demonstrated that ribavirin effectively induces the relocalization of nuclear eIF4E to the cytoplasm and the reduction of eIF4E as well as its target proteins, including suppression of Akt activation. This led to dramatic clinical improvement, including one complete remission, two partial remissions, two blast responses and four patients with stable disease. Final response data will be presented along with translational correlates. Notably, lack of response or relapse after remission was associated with lack of molecular response in leukemic blasts. Despite the encouraging responses of patient on ribavirin, all patients acquired resistance to therapy and eventually relapsed. Hence, we sought novel therapies to combine with ribavirin in order to overcome resistance and maintain remissions. Using a cell line that overexpresses eIF4E, we screened a library of 5000 known drugs and searched for compounds that synergize with ribavirin to suppress tumor growth. We identified nearly 50 lead compounds, many of which are structurally related, with similar biological activity, and are currently used medically for indications other than cancer. Early clinical observations suggest that combinations of cytotoxic agents lead to substantially better clinical outcomes relative to monotherapies. Furthermore, various drugs that suppress the PI3/Akt pathway were found to sensitize leukemia cells to Ara-C. Thus, we combined Ara-C with ribavirin in vitro, and observed an improved reduction in colony growth of AML specimens. Combination therapy with ribavirin and Ara-C in patients with acute myelocytic leukemia is currently ongoing. Preliminary results will be presented. Disclosures: Borden: Translational Therapeutics: Equity Ownership.

Description: The capacity of recombinant Hoxb4 protein to induce ex vivo expansion of HSCs identifies this protein as a potential HSC expanding factor. However, its short extra- and intra-cellular half-life (3–4 hours and 40–60 minutes, respectively) are hampering clinical applications of Hoxb4. The analyses of Hoxb4 molecular structure lead us to generate amino acid substitutions: Leu7→Ala, Tyr23→Ala and Tyr28→Ala in the Hoxb4 protein in order to decrease its degradation. Indeed, these modifications increased the intracellular stability of Hoxb4 protein ~3-fold compared to wild type Hoxb4 (Hoxb4(WT)). The ability of mutated Hoxb4 protein to favor expansion of hematopoietic progenitors was first examined in cultures initiated with 10% Hoxb4(WT)-GFP, 10% mutated Hoxb4-YFP expressing cells and 80% non-transduced cells. After an 18-day culture, the proportion of Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) cells increased to 50–60% in comparison to 30% for Hoxb4(WT) (p 〈 0.05), and no difference between the proliferation of Hoxb4(Tyr28→Ala) and Hoxb4(WT) cells could be identified. Western blot analyses showed that these Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) cells expressed ~ 4-fold higher and Hoxb4(Tyr28→Ala) cells ~ 8-fold lower levels of Hoxb4 protein than Hoxb4(WT) cells. The long-term reconstituting ability of these constructs was then evaluated in vivo using competitive repopulation assays. At 8 weeks after transplantation, Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) contributed to 11.5±2 and 13.1±1.8% of peripheral blood leukocytes (PBL) compared to 26.2± 4.3% determined for Hoxb4(WT), while after 16 weeks the progeny of Hoxb4(WT) cells generated the majority (≥65%) of the transplant-derived PBL in all recipients. Likewise, 16 weeks post transplantation Hoxb4 positive cells represented ≥80% of bone marrow, while cells expressing mutated Hoxb4 were present at ~10–12%level. Flow cytometry analysis of bone marrow, spleen and thymus revealed that mutated Hoxb4, like Hoxb4(WT) was expressed by all hematopoietic lineages, and that repopulation differences observed between mutated and WT Hoxb4 expressing cells were almost entirely attributable to myeloid lineage cells. However, short-term, non-competitive repopulation experiments showed that in the first 4 weeks post transplantation, mutated Hoxb4 expressing progenitors had a significantly greater contribution to the PBL recovery in comparison to Hoxb4(WT) (range 50–70% vs 16–30%, respectively; p 〈 0.05) for all three mutant proteins. Interestingly, this difference became less pronounced and non-significant after week 8 post transplantation. Together, these studies strongly suggest that different intracellular levels of Hoxb4 protein are affecting different types of hematopoietic progenitors. Early ex vivo expansion of clonogenic progenitors was achieved with mutated Hoxb4 proteins without impairing HSC long-term reconstituting ability. Thus, mutated Hoxb4 could represent a useful tool to accelerate engraftment after HSC transplantation.

Description: HOXB4 overexpression induces unique in vivo and in vitro expansion of hemopoietic stem cells (HSCs) without causing leukemia. Very little is known about the molecular basis underlying HOXB4-induced HSC self-renewal. We now report the in vitro proliferation and in vivo expansion capacity of primary bone marrow (BM) cells engineered to overexpress selected HOXB4 point mutants lacking either the capacity to directly bind DNA (HOXB4(A)), or to cooperate with members of the PBX family (HOXB4(W→G)) in DNA binding. The DNA binding–incompetent HOXB4 mutant failed to enhance the proliferation activity of transduced BM populations in vitro and HSC expansion in vivo. In contrast, the HOXB4(W→G) mutant conferred a pronounced in vitro proliferation advantage to the transduced BM populations, and dramatically enhanced their in vivo regenerative potential. We also demonstrate a correlation between HOXB4 protein levels and in vitro proliferative capacity of primary BM cells. Our observations thus suggest that the capacity of HOXB4 to induce HSC expansions is DNA-binding dependent and does not require direct HOX/PBX interaction, and sets the stage for identifying HOXB4-dependent targets involved in HSC expansion.

Description: The eukaryotic translation initiation factor eIF4E is elevated in 30% of malignancies including M4/M5 subtypes of acute myeloid leukemia (AML). The oncogenic potential of eIF4E arises from its ability to bind the 7-methyl guanosine (m7G) cap on mRNAs, thereby selectively enhancing eIF4E-dependent nuclear mRNA export and translation. We tested the clinical efficacy of targeting eIF4E in M4/M5 AML patients with a physical mimic of the m7G cap, ribavirin. Among 11 evaluable patients there were 1 complete remission (CR), 2 partial remissions (PRs), 2 blast responses (BRs), 4 stable diseases (SDs), and 2 progressive diseases (PDs). Ribavirin-induced relocalization of nuclear eIF4E to the cytoplasm and reduction of eIF4E levels were associated with clinical response. Lack of response or relapse coincided with continued or renewed nuclear localization of eIF4E. This first clinical study to target eIF4E in human malignancy demonstrates clinical activity and associated molecular responses in leukemic blasts. This trial is registered at ClinicalTrials.gov (NCT00559091).