Abstract 2539: Reactivating tumor suppression with small-molecule deregulators of the Rho-regulated diaphanous-related formin autoinhibitory mechanism

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Loss of all or part of the long-arm of chromosome 5 is a frequent karyotypic defect in myeloproliferitive neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To date, there is no clear mechanism that accounts for how any of the 5q tumor suppressor candidates contribute to hematopoietic stem cell growth control or how defects in their expression/function contribute to malignant progression. An attractive 5q- MDS tumor suppressor gene is DIAPH1 (5q31.3)]. DIAPH1 encodes the canonical mammalian Diaphanous-related (mDia) formin mDia1. Consistent with a role in tumor suppression, mDia1 knockout (KO) mice develop an age-dependent MDS-like phenotype. mDia1 is an effector for the Rho-family small GTP-binding protein RhoB, which is known to harbor tumor suppressor activity. mDia1 has a central role in the actin and microtubule dynamics that underlie cell division and directed migration. In this study we sought to determine if we could manipulate mDia activity to ‘reactivate’ tumor suppression by exploiting our working knowledge of the Rho-controlled mDia autoregulatory mechanism. GTP-bound Rho proteins activate mDia formins by binding to and disrupting an intramolecular autoinhibitory mechanism mediated by regulatory Dia-inhibitory (DID) and Dia-autoregulatory (DAD) domains that flank the conserved formin homology-2 (FH2) domain found in all formins that is responsible for assembling linear actin filaments and modulating microtubule stability. To identify small molecule activators of mDia formins, a screen for inhibitors of DID-DAD binding was conducted in a high-throughput format. We identified two structurally-related semi-thiocarbazones that bind within the DAD binding pocket of DID. These compounds, called Intramimics (IMM)-1 and −2, possess IC50 values for in vitro inhibition of DAD binding of 99 and 140 nM, respectively; the values were comparable to a DAD-derived peptide (280 nM). Consistent with mDia activation, exposure of fibroblasts and AML-derived cells leads to stabilization of actin and microtubule dynamics. The consequence is the induction of apoptosis, which was assayed by a variety of approaches. Using IMMs as tool compounds to explore tumor suppression mechanisms, IMM treatment activated serum response factor (SRF)-regulated gene expression. SRF is a growth factor-regulated transcription factor that also senses changes in actin assembly and is a potent activator of Egr1 expression. Egr1 is another 5q tumor suppressor candidate and is also a transcription factor that appears to be a master regulator of the well-known tumor suppressors PTEN and p53. Our next goal is to test IMMs in preclinical models for their ability to modulate myeloproliferation and to inhibit tumor growth, alone and in combination with other anti-tumor agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2539.