Acute myeloid leukemia (AML) is a subtype of leukemia which arises from the myeloid
cell lineage within the bone marrow. It is considered highly aggressive and difficult to treat,
with only approximately 55% of patients going on to live a disease-free life (DFL). With those
considered to have the most deleterious oncogenic mutations likely having no effective treatment
options resulting in durable remissions. This means that elucidating the genetic and molecular
basis of AML is crucial to the development of novel treatments for patients that have the
unfortunate diagnosis of AML. The Diacylglycerol (DAG) mimic, phorbol 12-myristate 13-
acetate (PMA) has been shown to induce cell senescence, macrophage differentiation, and
apoptosis in HL-60 cells over a 48-hour period. Despite PMA being too toxic as a therapeutic,
the data gained from understanding the molecular changes occurring in response to PMA could
help to develop targeted gene therapies for AML patients. Since DAG activates Protein Kinase C
(PKC), which induces the RAS/Raf pathway, it is expected that PMA would stimulate
proliferation through the same means. Thus, understanding how cell cycle arrest is being induced is of great interest. In addition, approximately 1250 genes change significantly in their
expression, suggesting of multiple mechanisms may lead to this proliferation block. The gene of
interest for this study is known as RASSF5. Through differential splicing and alternative
transcription start sites produces three isoforms of the gene. It was shown that during the PMA
response, the 5A and 5C isoforms are most abundant in the AML cell line HL-60. RASSF5A/C
cDNA clones were transfected into HL-60 cells and overexpression experiments successfully
showed a significant increase in expression on both the RNA and/or protein levels. With
overexpression confirmed, phenotypic changes were analyzed within the cells, and it was found
that 66% of 5A transfected cells were in senescence and 35% of 5C cells were also in
senescence. Furthermore, analysis of expression of putative downstream targets showed that the
likely pathway that is being interacted with by RASSF5A to induce senescence is the rb1
pathway (Figure 1). This implies that RASSF5A has an important role in promoting the
phenomenon known as oncogene-induced senescence in RAS-driven cancers such as AML, HL-
60 cells specifically.