Abstract 1939: NT219, a dual inhibitor of IRS1/2 and STAT3, suppresses cancer stem cell mediated resistance to KRASG12C and KRASG12D inhibitors in solid tumors
Authors: Reuveni Hadas , Al-Hallak Mohammed Najeeb , Motorwala Sarah , Beal Eliza , Kim Steve , Beydoun Rafic , Dyson Gergory , El-Rayes Bassel , Chen Herbert , Philip Philip A. , Shields Anthony F. , Pasche Boris , Sigalov Katy , Ben-David Hava , Miron Daphna , Azmi Asfar S. 
Published: March 2024

Abstract
Background NT219 is a novel small molecule, a dual inhibitor which uniquely triggers the degradation of Insulin Receptor Substrates 1/2 (IRS) and the dephosphorylation and suppression of STAT3, two major oncogenic targets that play a key role in drug resistance. Mutated KRAS (mKRAS) has long been referred to as undruggable until recently when KRASG12C inhibitors were approved. While KRASG12C is the most common oncogenic driver in NCSLC, KRASG12D is found in 42% of pancreatic tumors. We present NT219 suppressive effect on mKRASi resistant and sensitive cells of both PDAC and NSCLC, as well as on cancer stem cells (CSC) known to contribute to disease recurrence.
Method The inhibitory effects of NT219 as a monotherapy or in combination with either KRASG12C inhibitors (sotorasib/adagrasib) or KRASG12D inhibitor (MTRX1133) were tested in 2D cell growth and colony formation assays using mKRAS inhibitor sensitive and resistant cancer cell lines. The effect of NT219 and mKRASi on cancer stem cells was assessed using 3D spheroid assay evaluating spheroid size (microscopic measurements), cell viability (CellTiterGlo) and stemness marker expression (FACS analysis).
Results NT219 in combination with either sotorasib or MTRX1133 suppressed established spheroids of NSCLC(KRASG12C) and PDAC(KRASG12D) respectively, in terms of size, disintegration and viability. In NSCLC H358, spheroid satellites appeared following treatment with sotorasib, while the combination with NT219 suppressed it. CSC marker ALDH increased 7-fold in NSCLC spheroids as compared to 2D grown cells, while NT219 and sotorasib suppressed it. The expression of SOX2 stemness marker was reduced by NT219 alone (5 fold), unexpectedly induced by sotorasib (4 fold) and suppressed by NT219+sotorasib (15 fold vs sotorasib alone). In addition, significant induction of STAT3, was observed following sotorasib treatment and completely reversed by the addition of NT219. We also demonstrate that NT219 induces growth inhibition in both KRASiG12C resistant PDAC and NSCLC, and KRASiG12D resistant PDAC cells. NT219 exhibited synergistic effects with adagrasib and MTRX1133, in suppressing the proliferation of KRASG12C PDAC and NSCLC cells and KRASG12D PDAC cells, respectively. This combination showed a synergistic effect in xenograft model.
Conclusion We first showed the effect of NT219 in suppressing cancer stem cells, known to promote resistance and tumor recurrence. Synergistic effect of NT219 and mKRAS inhibitors, G12Cand G12D, in NSCLC and PDAC respectively, was demonstrated. Initial results suggest involvement of NT219 target proteins in resistance to mKRASG12C/D inhibitors. Treatment with sotorasib induced enhancement in stemness potential of mKRASG12C NSCLC, which NT219 efficiently suppressed, suggesting a novel therapy for this unmet need and a novel mechanism to combat resistance to mKRAS inhibitors
Citation Format: Hadas Reuveni, Mohammed Najeeb Al-Hallak, Sarah Motorwala, Eliza Beal, Steve Kim, Rafic Beydoun, Gergory Dyson, Bassel El-Rayes, Herbert Chen, Philip A. Philip, Anthony F. Shields, Boris Pasche, Katy Sigalov, Hava Ben-David, Daphna Miron, Asfar S. Azmi. NT219, a dual inhibitor of IRS1/2 and STAT3, suppresses cancer stem cell mediated resistance to KRASG12C and KRASG12D inhibitors in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1939.