Ink4a/ARF/Ink4b locus in Neurofibromatosis Type 1

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Summary/Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in patients with neurofibromatosis-1 (NF1). MPNSTs arise in NF1 patients from benign plexiform neurofibromas (PNFs) but it is unclear why only ~30% of PNFs transform into MPNSTs. Recent studies suggest that inactivation of the INK4a/ARF/INK4b locus (called CDKN2A for INK4a and ARF; CDKN2B for INK4b) generates a pre-malignant lesion called an atypical neurofibromatous neoplasm of uncertain biology (ANNUBP). ANNUBPs are the newly recognized precursor to MPNSTs, but mechanisms that drive and classify this transitional intermediate are poorly defined. INK4a/ARF/INK4b disruption is the main alteration currently linked to ANNUBPs, besides NF1 loss and RABL6A upregulation. In MPNSTs, the locus is altered at one, two or all three genes, with worse patient survival associated with loss of all three. Each gene (INK4a, ARF, and INK4b) encodes a tumor suppressor, but their separate contributions and cooperativity with other factors in driving cancer remain incompletely understood. Given their prominent role, a better understanding of INK4a, ARF, and INK4b in MPNST development is needed, as drugs targeting the locus are either approved or showing promise in other cancers. Our central hypothesis is p16Ink4a, ARF and p15Ink4b act cooperatively in multiple pathways to suppress the transformation of benign PNFs and ANNUBPs to MPNSTs. Aim 1 will define significant genetic and proteomic events coinciding with INK4a, ARF and INK4b inactivation in human ANNUBPs and MPNSTs by genetic, molecular and histologic analyses. Results will be correlated to clinical variables such as survival. The prognostic value of a newly developed liquid biopsy assay evaluating locus status and other tumor markers will be determined in NF1 patients. Aim 2 employs CRISPR editing of p16Ink4a, ARF and/or p15Ink4b in human PNF-derived cells to determine their roles in PNF-ANNUBP-MPNST transformation. Directed analyses of suspected MPNST driver genes in cells and mouse models will identify genes that selectively cooperate with INK4a, ARF, or INK4b loss to drive ANNUBP-MPNST transformation in vitro and in vivo. Aim 3 establishes the biological significance of drugs targeting Ink4a/Arf/Ink4b relevant pathways in PNF-ANNUBP-MPNST therapy and prevention. Predicted mediators of acquired resistance to therapy will be verified using molecular and pharmacologic approaches. Studies will determine the value of targeted therapy against pre-MPNST models to prevent malignant progression. Impact: Studies will provide new insights into INK4a/ARF/INK4b, one of the most frequently inactivated loci in human cancers. Innovative animal models of PNF-ANNUBP-MPNST progression will be generated and mechanisms of transformation leading to MPNST will be defined. Preclinical studies will assess a new combination therapy targeting Ink4a/Arf/Ink4b pathways in preventing malignant progression. Results will advance our understanding of events driving MPNST development, facilitating earlier diagnosis and pre-emptive interventions targeting ANNUBPs.