DNA and All-Trans Retinoic Acid as Immunotherapy or Add-on Adjuvants to 5-Azacytidine In Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis leading to cytopenia, which evolve to acute myeloid leukemia (AML). The ability of the DNA vaccination to induce effective immune responses has been demonstrated in different preclinical models of diseases. Having previously shown that DNA vaccination with a PMLRARaFrC plasmid in combination with all-trans retinoic acid (ATRA) induced long term remissions with appropriate immune responses in a mouse model of acute promyelocytic leukemia (APL), our aim was to use this strategy in a mouse model of MDS we have created using transgenic mice bearing a mutant NRAS and overexpression of human BCL-2.

We used our MDS-like triple transgenic mice bearing NRASD12/MMTVLTRtTA/TetoBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). We cloned part of the kanamycin resistance gene and adjacent pVax1 plasmid sequences flipped so the antisense sequences were inserted into the pVax1 vector (named Flipper). These sequences code for 5 polypeptides. We treated the MDS mice with either DNA alone, ATRA alone (10mg daily release for 21 days) or a combination of ATRA+DNA or as add-on therapy with 5-azacytidine (5-aza). 5-aza (5mg/kg) was administered 3 times a week and continued until death. Treatment with ATRA and/or DNA was initiated after 12 courses of 5-aza injections.

Sequencing of the Flipper insert predicted 5 peptides, which were synthesized and shown not to affect growth in vitro of myeloid leukemic NB4, K562 and p39 cell lines. In vivo injections of healthy mice with these peptides had no effect on mortality. The inserted sequences did not appear to have detrimental effects. The studies on our MDS mouse model show that DNA vaccination, alone or in combination with ATRA induces a long-term survival of the treated mice compared to untreated controls (p<0,0001). DNA vaccination results in an increase of interferon-gamma secretion as measured by ELISPOT, increased CD4+/CD44^hi/CD62-L^lo memory T cells in the peripheral blood and maintains stable disease as determined by the persistence of low peripheral blood platelet counts and the of the Mac-1hi/GR-1^lo populations, which mark the primitive cells and the ERK phosphorylation signatures measured by the NanoPro (Cell Biosciences). Memory T-cell levels increased 3-fold (at 6% compared to 2% in healthy controls) and remained high. The DNA vaccine stabilized the disease and its protective effect may persist for up to 10 months with increased expression of MYD88, which is downstream of toll-like receptors, in long-term survivors, suggesting the activation of this DNA pathway. ATRA alone also had efficacy in this model. As there was no evidence of differentiation as the Mac-1^hi/Gr-1^lo blast cell population remained unchanged after treatment compared to the levels before treatment, it is possible that in this setting where the leukemic stem cells are pro-apoptotic, that ATRA is acting as an immuno-adjuvant and the antigen shedding that occurs allows cross presentation and induces immune responses potentiated by ATRA. However, the disease in this group progresses and the mice die earlier than the other treatment groups (mean survival of 4.5 months compared with the untreated group of 1 month, p=0.0016). Vaccination with an adjuvant DNA alone or in combination with ATRA, results in a significant extension of lifespan in MDS mice. As add-on therapy to 5-aza the response rate increases from 20% with 5-aza alone to 50% with either ATRA or DNA and 100% with the 5-aza, ATRA and DNA combination. These studies suggest that adjuvant therapy with ATRA+DNA may have a role in addition to conventional therapy in prolonging remissions and may be promising for clinical trials.

Fenaux:Novartis, Janssen, Cilag, Roche, Amgem, GSK, Merck and Cephalon: Honoraria, Research Funding. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Padua:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.