Abstract
Abstract Background: AML is phenotypically diverse. However, genome-wide sequencing studies indicate that median number of non-synonymous mutations in AML is 8 (B Vogelstein Science 2013) and that the same pathways are affected in tumors with distinct genetic alterations. These insights provided the impetus to confirm and extend the previously published observation that immunization with normal human white blood cells (WBC) whose surface charge had been modified in vitro by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross-reacted against a broad range of leukemias (Nature 232:197-198,1971). Specific Aims: 1) Isolation and molecular characterization of a shared antigenic moiety from human AML, 2) Examine the prevalence- and role in AML of IQGAP1, which was identified as a shared antigenic moiety. Methods: WBCs from healthy donors were incubated with FDNB at 104 molecules/cell for 12-15 minutes in PBS. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells. After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested for their ability to stain AML cell lines by flow cytometry and clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was done using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immuneprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined from raw data from Gene Expression Omnibus at the NCBI using Partek Genomic Suite. IQGAP1 expression was knocked down by shRNA and the effect on proliferation and colony formation was measured. Results: Sera from experimental rabbits stained AML cell lines with greater intensity by flow cytometry compared to serum from the control rabbit. Western blotting of whole cell lysates of clinical AML samples revealed bands that were recognized by immune serum from experimental rabbits but not the control rabbit. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples revealed IQGAP1 as being differentially recognized in independent experiments. Western blots of human AML samples probed with anti-IQGAP1 antibody revealed the predicted 190 kDa band. The fold change in IQGAP1 expression in normal bone marrow versus AML was -3.22636, p-value 2.62 × 10e-7. Knocking down expression of IQGAP1 in K562, MV4-11 and THP1 cell lines resulted in significant decrease in proliferation and colony formation. Conclusion and Future Directions: IQGAP1 was identified as a shared antigenic moiety in. IQGAP1 is over-expressed in AML compared to normal bone marrow. Knocking down IQGAP1 expression in AML cell lines decreased proliferation and colony formation. Experiments to determine the mechanistic basis of the effect of FDNB on cells and if IQGAP1 is “druggable” are underway. Citation Format: Deepak M. Sahasrabudhe, Jeremy Bechelli, Fred P. Hagen, Mark Paris, Marlene Balys, Mohammad Minhajuddin, Jane Liesveld. IQGAP1 in human acutae myelogenous leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2015-4041
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