Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biochemistry Program

Abstract

Mixed lineage leukemias (MLL) are a group of acute and aggressive leukemias. They account for over 70% of infant leukemias, and 10% of acute adult leukemias. Pediatric ALL and therapy related MLL leukemias carry poor prognosis in spite of several advancement in the field of leukemia research. Therefore, new therapies for MLL leukemias are needed.

Majority of MLL leukemias arise due to the balanced translocations of the MLL gene. As a result of these translocations, chimeric MLL fusion proteins are expressed. The most frequently occurring MLL fusion proteins are known to aberrantly recruit the super elongation complex (SEC) resulting in constitutive transcription of genes that promote the development of leukemia. Hence, our strategy is to target the SEC as a means of inhibiting MLL leukemia. AF4 and AF9 proteins co-purify with components of the SEC and directly interact with each other. Our laboratory has previously identified the domain of AF4 which is required for AF9 interaction and demonstrated that inhibition of this interaction using an AF4 mimetic peptide results in decreased viability of leukemia cell lines expressing MLL fusion genes. The AF4 mimetic peptide was modified to improve its in vivo stability and the newly designed peptide was designated SPK111.

Here, we demonstrate that SPK111 peptide inhibits the AF4-AF9 interaction and reduces the activity of the SEC using luciferase reporter assays. Further, we show that SPK111 selectively reduces the viability of MLL leukemic cells in vitro. It induces membrane permeability and necrotic cell death. In order to test the in vivo efficacy of SPK111, we generated mice xenografts of MOLM13 and KOPN8 MLL leukemia cells. We observed a trend toward prolonged survival of xenografted mice following SPK111 treatment. However, the increased survival of treated mice did not reach statistical significance. A larger dose or dosing at an earlier point in time during disease progression had little effect on survival. Although it was difficult to achieve efficacy in vivo, pretreatment of leukemic cells with SPK111 prior to tail vein injection effectively inhibited xenograft establishment. This suggests that SPK111 is effective on leukemia initiating cells and may be developed as an effective bone marrow purging agent. We also developed an ELISA for detection of serum SPK111 which can be used for future kinetic studies.

PFWT is an AF4 mimetic peptide similar to SPK111. Previous studies suggest that PFWT perturbs the actin cytoskeleton which is likely to induce cell death. However our investigations show that PFWT does not adversely affect the filamentous actin content of leukemic cells. Moreover, pretreatment with actin stabilizing drugs does not protect against PFWT induced cell death. An apparent 10 kDa increase in the molecular weight of the AF9 protein was identified on exposure to PFWT. Our analysis of probable post-translational modifications shows the absence of O-glycosylation and monoubiqutination. Interestingly, multiple phosphorylation sites and an acetylation site of AF9 were identified using mass spectroscopy.

Our studies on the AF4 mimetic peptide, suggest that inhibition of the AF4-AF9 protein-protein interaction serves as an effective therapy for MLL leukemias.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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