Tuberous sclerosis complex (TSC) is a familial tumor disorder for which there is no effective medical therapy. Disease-causing mutations in the TSC1 or TSC2 gene lead to increased mammalian target of rapamycin (mTOR) kinase activity in the conserved mTOR signaling pathway, which regulates nutrient uptake, cell growth, and protein translation. The normal function of TSC1 and TSC2 gene products is to form a complex that reduces mTOR kinase activity. Thus, mTOR kinase inhibition may be a useful targeted therapeutic approach. Elevated interferon-gamma (IFN-gamma) expression is associated with decreased severity of kidney tumors in TSC patients and mouse models; therefore, IFN-gamma also has therapeutic potential. We studied cohorts of Tsc2+/- mice and a novel mouse model of Tsc2-null tumors in order to evaluate the efficacy of targeted therapy for TSC. We found that treatment with either an mTOR kinase inhibitor (CCI-779, a rapamycin analog) or with IFN-gamma reduced the severity of TSC-related disease without significant toxicity. These results constitute definitive preclinical data that justify proceeding with clinical trials using these agents in selected patients with TSC and related disorders.

Tuberous sclerosis is a hamartoma syndrome due to mutations in TSC1 or TSC2 in which cardiac rhabdomyomas are seen in approximately 60% of patients. These lesions have an unusual natural history as they are usually most prominent immediately after birth and spontaneously resolve in most cases. To develop a mouse model of this lesion, we used a conditional, floxed allele of Tsc1 and a modified myosin light chain 2v allele in which cre recombinase expression occurs in ventricular myocytes. Mice with ventricular loss of Tsc1 had a median survival of 6 months and developed a dilated cardiomyopathy with the occurrence of scattered foci of enlarged ventricular myocytes. The enlarged cells were periodic acid-Schiff positive indicating the presence of excess glycogen and expressed elevated levels of phospho-S6, similar to findings in patient rhabdomyoma cells. The observations confirm that rhabdomyomas occur through a two hit mechanism of pathogenesis. However, the mice showed no evidence of fetal/neonatal demise, and there was no evidence of proliferation in the lesions. We propose that these differences are due to the timing of loss of Tsc1 in the ventricular myocytes and/or the truncated gestational period in the mouse compared with humans, during which progestational hormones may accentuate the growth of patient rhabdomyomas.

ABSTRACT

The inhibition of Aurora kinases in order to arrest mitosis and subsequently inhibit tumor growth via apoptosis of proliferating cells has generated significant discussion within the literature. We report a novel class of Aurora kinase inhibitors based upon a phthalazinone pyrazole scaffold. The development of the phthalazinone template resulted in a potent Aurora-A selective series of compounds (typically >1000-fold selectivity over Aurora-B) that display good pharmacological profiles with significantly improved oral bioavailability compared to the well studied Aurora inhibitor VX-680.

Pulmonary lymphangioleiomyomatosis and abdominal angiomyolipoma are related lesions for which there is no authentic animal model. Both of these proliferative lesions occur in sporadic patients, and at much higher frequency in patients with tuberous sclerosis, which is due to mutations in the TSC1 and TSC2 genes. Tsc1 +/À À and Tsc2 +/À À mice frequently develop liver hemangioma. We found that the Tsc mouse liver hemangioma are composed predominantly of endothelial cells but with a smooth muscle component, and express HMB45 antigen, estrogen receptor, and progesterone receptor, similar to lymphangioleiomyomatosis and angiomyolipoma. Estrogen treatment significantly accelerated the development of liver hemangioma in Tsc1 +/ À À female mice, with 91% having liver hemangioma and 55% having severe lesions by 7 months of age. Similarly, an increased frequency and severity of liver hemangiomas was seen in Tsc1 +/ À À males treated with estrogen. In contrast, tamoxifen treatment for 9 months significantly reduced the frequency and severity of hemangiomas in Tsc1 +/ À À female mice. In addition, estrogen treatment significantly increased serum vascular endothelial growth factor levels in Tsc1 +/ À À mice, whereas tamoxifen reduced vascular endothelial growth factor levels. These mouse model observations indicate the importance of estrogen signaling in vivo for the growth of tuberous sclerosis lesions, suggesting the possible benefits of tamoxifen therapy for the treatment of angiomyolipoma and lymphangioleiomyomatosis. (Cancer Res 2005; 65(6): 2474-81)

No abstract

Redx Oncology has developed novel, differentiated, reversible small molecule inhibitors of BTK, combining current best-in-class potency with improved selectivity profiles, which are suitable for oral, once daily dosing, designed to be equipotent against wild-type and C481S BTK. BTK is a member of the src-related Tec family of cytoplasmic tyrosine kinases. BTK plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK inhibitors have therefore been developed with the aim of treating B-cell malignancies that are dependent on BCR signaling, such as CLL and NHL. Ibrutinib is an irreversible BTK inhibitor that has been approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors specifically target a cysteine residue C481 within BTK. Following treatment with ibrutinib, cases of secondary resistance have emerged in both CLL and MCL patients. Acquired mutations within BTK such as C481S, C481Y, C481R, C481F have been reported in the literature and clearly interfere with covalent drug binding. It has been predicted that the incidence of observed resistance will increase as clinical use outside clinical trials expands over time. Here, we present our reversible BTK inhibitor series demonstrating subnanomolar binding affinity for WT and C481S forms of BTK, that inhibits formation of pBTK in both WT and C481S BTK expressing cells. In addition, the compound demonstrated significant in vitro potency against a number of lymphoma cell lines including inhibition of BCR signaling and proliferation in OCI-Ly10 cells at nanomolar concentrations. To further investigate the binding nature of these compounds, PBMC wash out studies, measuring CD69 as a marker of B-cell activation, were used to highlight the reversible activity of these compounds. Some BTK inhibitors also inhibit ITK, which plays a critical role in FcR-stimulated NK cell function that is required for ADCC. ADCC is the mechanism that anti-CD20 antibodies, such as rituximab, are believed to activate, and ibrutinib has been shown to antagonize this mechanism in vitro. As rituximab-combination chemotherapy is today's standard of care in B-cell malignancies, it would be desirable to have a BTK inhibitor with high selectivity for BTK over ITK. At 1 μM, our lead compound is highly selective when tested against 469 kinase and did not show significant inhibition against other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, the compound has high selectivity versus structurally related cysteine-containing kinases (including EGFR and ITK) both in enzyme and cellular assays. Our lead compound has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile to competitor compounds. In vivo PK demonstrated good oral bioavailability and in vivo efficacy has been demonstrated. Citation Format: Nicolas E S Guisot, Victoria Walker, Stuart A. Best, Valentina Abet, Rose Chappell, Juliette Emmerich, Kelvin Ho, James R. Kelly, Kristina Lyons, Melanie Müller, Julienne Refuerzo, Louise Sargent, Fatima Talab, Madelene Waldron, Matilda Bingham, Mary-Ann Campbell, Caroline Phillips, Richard Armer. Development of novel, selective, reversible inhibitors equipotent against wild-type and C481S Bruton's tyrosine kinase (BTK). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4795.

The B-cell receptor (BCR) signaling pathway is required for the survival, activation, proliferation and differentiation of B-cells. Bruton's Tyrosine Kinase (BTK) is a member of the Tec protein tyrosine kinase family that has emerged as an attractive target for the treatment of B-cell malignancies due to the critical role it plays in BCR signaling. Redx Oncology has developed novel differentiated small molecule inhibitors of BTK, combining current best-in-class potency with distinct selectivity profiles, which are suitable for oral once daily dosing. Here we present REDX05194, the result of a successful lead optimization of our proprietary BTK inhibitor series. REDX05194 is a highly selective, covalent BTK inhibitor displaying subnanomolar binding affinity for BTK (0.39 nM) and nanomolar potency towards BTK in a biochemical assay (3.67 nM). In cell proliferation assays, REDX05194 showed significant in vitro potency against ABC-DLBCL cell lines inhibiting the growth of both TMD-8 (0.89 nM) and OCI-Ly10 (1.36 nM) cells. Analysis of BCR signaling in several lymphoma cell lines, including cell lines of ABC-DLBCL, MCL and FL origin, revealed that treatment with REDX05194 inhibits BTK autophosphorylation and downstream activation of PLCγ2. In human PBMCs, REDX05194 inhibited anti-IgM stimulated upregulation of the CD69 activation marker in CD19 positive B-cells. In addition, using a fluorescent probe that binds to BTK, occupancy of BTK in PBMCs has been demonstrated in response to increasing concentrations of REDX05194. To assess selectivity, 456 kinases were screened at 1 μM, confirming that REDX05194 does not significantly inhibit other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, REDX05194 was shown to have high selectivity versus structurally related cysteine-containing kinases such as ITK in binding assays, and EGFR as demonstrated in both binding and cellular assays. REDX05194 also has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile and solubility compared to competitor compounds. REDX05194 demonstrated in vivo efficacy in a mouse collagen-induced arthritis (CIA) model. At 10 mg/kg and 30 mg/kg QD, REDX05194 significantly improved all clinical readouts, including disease severity, compared to the vehicle group. Histological data showed that approximately 1/3 of the mice had no or minimal pannus infiltration and no bone resorption, or had bone resorption restricted to small areas. These findings demonstrate potential clinical efficacy and a dose response. In conclusion, REDX05194 is a highly selective and potent BTK inhibitor with proven efficacy in several lymphoma cell lines and human PBMCs and in vivo efficacy demonstrated in a mouse CIA model. Disclosures No relevant conflicts of interest to declare.