ABSTRACT Tumor suppressor p53 is the quintessential guardian of the genome whose function is inhibited in greater than 50% of all human cancers. Though mutation and deletion of p53 are major contributors to p53 inactivation, overexpression of the negative regulators MDM2 and MDM4 (MDMX) are also known to inactivate p53, thus leading to the cancer phenotype. Our lab has shown that specific types of cell stress initiate the generation of an alternatively spliced isoform of MDM2. The predominant MDM2 alternative isoform, MDM2-ALT1 also known as MDM2-B, functions to primarily activate the p53 pathway by inhibiting MDM2 and MDM4 in a dominant negative fashion. Paradoxically, this isoform is upregulated in several human cancers, such as pediatric high-grade gliomas, astrocytomas, rhabdomyosarcomas (RMS), and liposarcomas, as well as adult cancers such as lymphomas and those of the breast. Thus, MDM2-ALT1 plays opposing roles in cancer progression dependent upon the context of its expression. In the proposed research, we will study the underpinnings of the control of the p53 pathway by MDM2-ALT1 to better understand 1) the specific mechanism by which that MDM2-ALT1 is generated in cancer and 2) the ability of the resultant isoforms to be targeted using splice-switching oligonucleotides. We hypothesize that the expression of oncogenic MDM2- ALT1 is modulated by alterations in protein and RNA nuclear factors during the progression to tumorigenesis and can be targeted to induce splicing changes. We will use assays that identify and measure splice regulation in conjunction with gene editing approaches to identify RNA sequences and their respective nuclear factor- binding partners necessary for regulation of MDM2 splicing. Furthermore, we will use novel genetically engineered mouse models as well as established mouse xenograft assays and novel splice switching oligonucleotides (SSOs) to modulate MDM2 isoform levels. Our work will broaden our knowledge of combinatorial regulation of RNA processing in response to stress and in cancer and interrogate the utility of MDM2 isoforms modulation for rational control of the p53 pathway.