Abstract 1397: A phenotypic-based drug discovery approach against tumors with MTAP loss

The Methylthioadenosine phosphorylase (MTAP) gene is in chromosome 9q21 location, which includes p16/CDKNA and is homozygously deleted in about 15% of all cancers, including a large proportion of gliomas, mesotheliomas and T cell lymphomas. MTAP cleaves methylthioadenosine (MTA), a byproduct of polyamine synthesis, to regenerate methionine and adenine. Several functional genomics screens have identified the downstream genes MAT2A and PRMT5 as synthetically lethal with MTAP loss. These two targets have been the subject of several drug discovery efforts. Our approach was based on a forward chemical genetic approach, in which we employed two isogenic cell lines, PANC1, with MTAP loss and PAM cells where MTAP is re-introduced. A library of over 200,000 compounds was screened for hits that affected the viability of PANC1 cell, whereas spared PAM cells. One hit series was found to reproducibly inhibit the proliferation of PANC1 cells with EC50s in the low single digit uM range and one order of selectivity over PAM cells. Phenotype-based optimization resulted in improved analogs, with prototype MTB-22252 exhibiting an EC50 of 47nM in PANC1 cells and 15-fold selectivity. An affinity probe was designed based on an analog of MTB-22252 bearing a biotin moiety via a linker and an azide group for photoactivatable cross-linking with potential interacting proteins. This probe maintained cellular activity and selectivity and it was used in an MS-proteomics experiment in PANC1 cells. The top protein identified by this experiment showed high enrichment over DMSO control and was effectively competed out by MTB-22252. MTB2252 and analogs thermo-stabilized this protein by up to 50C in PANC1 cells. PANC1 cells expressed high levels of this protein, whereas insensitive PAM cell did not express it at all. In additional cell line pairs, sensitivity to MTB-2252 correlated well with the expression levels of this protein. Reintroduction of MTAP in cells lacking MTAP and this new protein, resulted in loss of expression of the new protein, suggesting a regulatory connection. In a wider cell panel of over 130 cell lines, there was correlation of sensitivity with MTAP loss and even better correlation with the expression levels of this protein. Some of the most sensitive cell lines included several colorectal, glioma and hematologic cancer cell lines. In vivo evaluation of MTB-9655 against some of the most sensitive tumor cell lines is on-going. Citation Format: Andreas Goutopoulos, Iris