Over Expression of MN1 Accelerates Leukemia Onset and Confers Resistance to Chemotherapy by Suppression of p53 and Bim

Abstract 2501

Acute myeloid leukemia (AML) is an aggressive malignancy of immature myeloid precursors that leads to progressive marrow failure and death. This disease will affect approximately 12,950 people this year in the United States, causing 9,050 deaths. The most common treatment is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease.

AML is a genetically diverse malignancy and karyotype can be used to delineate prognosis. There is a clear link between chromosomal abnormalities and resistance to chemotherapy as complete remission rates are significantly different between groups. Additionally, there are now multiple submicroscopic genetic alterations that have been found to effect prognosis. These alterations can be mutations, over or under expression of a particular gene.

MN1 is a transcription co-factor and several studies have demonstrated its over-expression confers a worse prognosis. High MN1 expressers were less likely to achieve a remission and had lower 3 year survival rates. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. The effect of MN1 on response to standard chemotherapy is currently unknown.

To determine the effect of MN1 expression on therapy response we infected murine MLL-ENL driven AML cells with retroviral vectors that expressed MN1. When partially infected populations were exposed to a titration of either Ara-C or doxorubicin MN1 expressing cells were significantly enriched compared to untreated controls. When cells were exposed to a titration of Ara-C the MN1 expressing cells were enriched up to1.69 fold and when exposed to doxorubicin were enriched up to 3.80 fold. Both results were highly statistically significant with p values of 0.004 and < 0.0001. Consistent results were obtained with repeated infections and with separately derived MLL-ENL lines. Additionally, MN1 was able to confer therapy resistance to anthracycline resistant Flt3-ITD expressing cells suggesting non-overlapping mechanisms. Purified populations of cells expressing MN1 were resistant to Ara-C when compared to the parental leukemia (IC₅₀ 175.6nM vs 67.28nM) and highly resistant to doxorubicin. Consistent with these results human OCI-AML3 cells expressing MN1were enriched by 1.6 fold when exposed to doxorubicin, a highly significant result with a p value of 0.0002. In contrast a control vector without MN1 was not significantly enriched.

In vivo when mixed leukemia cells were injected into syngeniec recipients MN1 expressers were significantly enriched in the femoral bone marrow of treated animals compared to controls. Treated animals had 90.58% (+/−0.66) MN1 expressing blasts compared to 55.38% (+/−5.25) in controls. This result was highly statistically significant with a p value of < 0.0001. This observation was reproducible in a separately derived MLL-ENL driven cell line. Additionally, the engraftment of MLL-ENL and Flt3-ITD expressing cells was significantly increased by MN1 expression leading to shorter survival in recipient animals despite the already highly aggressive nature of the parental leukemia.

When MN1 expressing cells were exposed to doxorubicin or Ara-C they displayed significantly lower Annexin V positivity consistent with an attenuated apoptotic response (3.65 vs 34.79, p=<0.0001). When we examined BH3 only family member induction following exposure to Ara-C and doxorubicin we found significantly decreased levels of Bim induction by QPCR in cells expressing MN1. Similarly, stabilization of p53 following treatment was blunted in MN1 expressers as was induction of its downstream targets p21 and MDM2. Importantly the amount of DNA damage induced by doxorubicin as assessed by γH2AX foci was not different between MN1 expressing cells and the parental leukemia.

These data suggest that over expression of MN1 confers resistance to both Ara-C and doxorubicin in vitro and in vivo by suppression of Bim induction and p53 response. These observations suggest a biological explanation for the clinical observation that it confers a worse prognosis.