Session: 602. Myeloid Oncogenesis: Basic: Poster I
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Acute Myeloid Malignancies, AML, Bone Marrow Failure Syndromes, Translational Research, Combination therapy, hematopoiesis, Diseases, Therapies, Myeloid Malignancies, Biological Processes, molecular biology, Study Population, Animal model
To address this possibility in primary leukemia cells, we generated a new knock-in mouse model with the CM fusion gene flanked by loxP sites (CbfbflMYH11). This allows for the fusion gene to be deleted by Cre Recombinase (Cre) after leukemia development. To test the effect of loss of CM in vitro, we transduced Cbfb+/flMYH11 leukemia cells from three independent CbfbflMYH11 mice with a lentivirus expressing Cre. We confirmed the deletion of CM by qPCR and western blot. In Cbfb+/flMYH11, Cre+ cells, we observed a statistically significant increase in Annexin V staining and a decrease in colony forming ability as compared to control Cbfb+/flMYH11 cells. To determine if the colonies that did grow from Cre-infected cells had successfully deleted CM, individual colonies were picked. We found that majority of the colonies retained the unexcised CM allele, but 5-20% of colonies had successfully deleted CM, indicating that a rare population of leukemia cells can survive without CM in vitro.
To test the requirement for CM in vivo, we used an inducible shRNA against CM rather than Cre+ CbfbflMYH11 leukemia cells, as the inducible knockdown model will allow us to avoid potential defects in engraftment. We transduced CM expressing leukemic cells with a lentiviral construct with a doxycycline (Dox) inducible shRNA against MYH11 (shMYH11) that also expresses GFP. To test the feasibility of this approach in vitro, the sorted shCM+ leukemia cells were incubated with Dox for 48 hours. We found that Cbfb-MYH11 mRNA and protein were decreased in Dox treated leukemia cells, as compared to control cells. In addition, Dox treated shCM+ cells showed increased apoptosis and decreased colony forming ability, similar to Cbfb+/flMYH11 Cre+ cells. To test the effect of CM knockdown in vivo, shCM transduced leukemia cells were transplanted into sub-lethally irradiated mice and monitored for leukemic engraftment. Once significant leukemic burden in the peripheral blood was detected, mice were given Dox or vehicle in their drinking water. Starting on day 7, we observed a statistically significant decrease in leukemia burden in Dox treated mice in the peripheral blood and spleen, with the percent of leukemia cells below 5% starting on day 14. In the bone marrow, there was a statistically significant decrease in leukemic burden starting on day 14, but the leukemia cells remained at 30-40% through day 28. Our initial tests indicate that the remaining GFP+ leukemia cells in the bone marrow express reduced levels of CM, implying that these cells may not strictly require the fusion protein or may be able to survive with only low levels of Cbfb-MYH11 activity. In a separate, small cohort of shCM+ transplanted mice, we treated the mice with Dox or control water and monitored for leukemia development. All the control treated mice developed fatal leukemia by day 21, whereas the majority of the Dox treated mice survived until day 42 of treatment. Interestingly, among the 6 Dox treated mice, 2 showed re-emergence of GFP+ leukemia cells in the peripheral blood on day 21 and 28, respectively. We are currently testing whether these relapsed leukemia cells re-express CM.
Taken together, our results indicate that Cbfb-MYH11 is largely required for leukemia maintenance, although a subset of cells may be able to survive without the fusion gene. These results imply that drugs targeting the Cbfb-MYH11 have the potential to be effective treatments for inv(16) AML, although substantial inhibition of the fusion protein will likely be needed for effective treatment of the disease.
Disclosures: No relevant conflicts of interest to declare.
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