Masters Thesis

Novel antagonists of the SH2 domain of GRB2 decrease chronic myeloid leukemia (CML) cell proliferation via the JAK/STAT pathway

Chronic myeloid leukemia (CML) is a disease that affects the normal growth of myeloid cells, which are blood cells that protect the body against foreign invaders in the blood and bone marrow. In humans, 95% of CML cases are caused by a chromosomal translocation that inappropriately links the breakpoint cluster region (BCR) to Abelson murine leukemia viral oncogene-1 (ABL1), forming a mutant oncogene called BCR-ABL1 . A protein that physically interacts with BCR-ABL1 is growth receptor bound protein-2 (GRB2), an intracellular adaptor protein involved in cell growth and differentiation. Specifically, BCR-ABL1 binds to a region of GRB2 known as the SRC homology-2 (SH2) domain. This interaction transforms hematopoietic stem and progenitor cells, initiating leukemic transformation. The current frontline therapy to treat CML is a tyrosine kinase inhibitor, imatinib. Some patients have developed a resistance to imatinib, and thus the demand for additional anticancer drugs are needed. To prevent growth of CML cells, Dr. Arpin and her students of the CSU Chemistry and Biochemistry department created two novel SH2 antagonists (NHD2-15 and NHD2-114) and we tested their ability to prevent cell proliferation in the human BCR-ABL1 + K562 myelogenous leukemia cell line. The most significant growth reduction was observed 72 hours after the addition of 30 μM of NHD2 - 15. Furthermore, adding drugs combinatorially (60 μM NHD2-15, 30 μM NHD2-114, and 1 μM imatinib) to K562 cells showed over 2-fold growth reduction than with imatinib alone. To assess if these compounds are toxic to living organisms, we added the two compounds individually to the water of healthy adult zebrafish, and found that NHD2-15 was non-toxic. After using an enzyme-linked immunosorbent assay (ELISA) we also found that these two novel drugs exhibited prominent binding affinities to GRB2; NHD2-15 with K d = 119 ± 2 μM, and NHD2 - 114 with K d = 440 ± 7 μM (Lewis et al., in revision, 2019). Western blots were performed to determine the pathway these novel antagonists in fluence, and to ultimately indicate if these drugs stop cancerous cell proliferation via the Janus kinase signal transducer and activators of transcription (JAK/STAT) pathway, as well as the mitogen-activated protein kinase and phosphoinositide 3-kinase (M APK/PI3K) pathway. Results of western blotting indicated that the combinatorial treatments of both novel drugs reduces expression of proteins involved in both the JAK/STAT and MAPK/PI3K pathways, suggesting these compounds inhibit different target proteins within BCR-ABL+ cells to decrease leukemic cell proliferation. This research should provide an additional alternative treatment for patients who develop imatinib resistance.

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