kx-01 and Breast-Neoplasms

Application of tumor genome sequencing has identified numerous loss-of-function alterations in cancer cells. While these alterations are difficult to target using direct interventions, they may be attacked with the help of the synthetic lethality (SL) approach. In this approach, inhibition of one gene causes lethality only when another gene is also completely or partially inactivated. The EPHB6 receptor tyrosine kinase has been shown to have anti-malignant properties and to be downregulated in multiple cancers, which makes it a very attractive target for SL applications. In our work, we used a genome-wide SL screen combined with expression and interaction network analyses, and identified the SRC kinase as a SL partner of EPHB6 in triple-negative breast cancer (TNBC) cells. Our experiments also reveal that this SL interaction can be targeted by small molecule SRC inhibitors, SU6656 and KX2-391, and can be used to improve elimination of human TNBC tumors in a xenograft model. Our observations are of potential practical importance, since TNBC is an aggressive heterogeneous malignancy with a very high rate of patient mortality due to the lack of targeted therapies, and our work indicates that FDA-approved SRC inhibitors may potentially be used in a personalized manner for treating patients with EPHB6-deficient TNBC. Our findings are also of a general interest, as EPHB6 is downregulated in multiple malignancies and our data serve as a proof of principle that EPHB6 deficiency may be targeted by small molecule inhibitors in the SL approach.

Topics: Acetamides; Animals; Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Membrane; Female; Fluorescent Dyes; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Immunohistochemistry; Indoles; Mice; Mice, Inbred NOD; Mice, SCID; Morpholines; Pyridines; Receptors, Eph Family; RNA, Small Interfering; src-Family Kinases; Sulfonamides; Synthetic Lethal Mutations; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Src kinase is elevated in breast tumors that are ER/PR negative and do not overexpress HER2, but clinical trials with Src inhibitors have shown little activity. The present study evaluated preclinical efficacy of a novel peptidomimetic compound, KX-01 (KX2-391), that exhibits dual action as an Src and pretubulin inhibitor. KX-01 was evaluated as a single-agent and in combination with paclitaxel in MDA-MB-231, MDA-MB-157, and MDA-MB-468 human ER/PR/HER2-negative breast cancer cells. Treatments were evaluated by growth/apoptosis, isobologram analysis, migration/invasion assays, tumor xenograft volume, metastasis, and measurement of Src, focal adhesion kinase (FAK), microtubules, Ki67, and microvessel density. KX-01 inhibited cell growth in vitro and in combination with paclitaxel resulted in synergistic growth inhibition. KX-01 resulted in a dose-dependent inhibition of MDA-MB-231 and MDA-MB-157 tumor xenografts (1 and 5 mg/kg, twice daily). KX-01 inhibited activity of Src and downstream mediator FAK in tumors that was coincident with reduced proliferation and angiogenesis and increased apoptosis. KX01 also resulted in microtubule disruption in tumors. Combination of KX-01 with paclitaxel resulted in significant regression of MDA-MB-231 tumors and reduced metastasis to mouse lung and liver. KX-01 is a potently active Src/pretubulin inhibitor that inhibits breast tumor growth and metastasis. As ER/PR/HER2-negative patients are candidates for paclitaxel therapy, combination with KX-01 may potentiate antitumor efficacy in management of this aggressive breast cancer subtype.

Topics: Acetamides; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dasatinib; Drug Synergism; Estrogen Receptor alpha; Female; Focal Adhesion Kinase 1; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neoplasm Invasiveness; Neoplasm Micrometastasis; Neovascularization, Pathologic; Paclitaxel; Peptidomimetics; Pyridines; Pyrimidines; Receptor, ErbB-2; Receptors, Progesterone; src-Family Kinases; Thiazoles; Tubulin Modulators; Tumor Burden; Xenograft Model Antitumor Assays

KX2-391 (KX-01/Kinex Pharmaceuticals), N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetamide, is a highly selective Src substrate binding site inhibitor. To understand better the role of pyridine ring and N-benzylsubstitution in KX2-391 and establish the structure-activity relationship, a number of N-benzyl substituted (((2-morpholinoethoxy)phenyl)thiazol-4-yl)acetamide derivatives containing thiazole instead of pyridine were synthesized and evaluated for Src kinase inhibitory activities. The unsubstituted N-benzyl derivative (8a) showed the inhibition of c-Src kinase with GI(50) values of 1.34 μM and 2.30 μM in NIH3T3/c-Src527F and SYF/c-Src527F cells, respectively. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), breast carcinoma (BT-20), and leukemia (CCRF-CEM) cells. 4-Fluorobenzylthiazolyl derivative 8b exhibited 64-71% inhibition in the cell proliferation of BT-20 and CCRF cells at concentration of 50 μM.

Topics: Acetamides; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Female; Humans; Inhibitory Concentration 50; Leukemia; Mice; Morpholines; NIH 3T3 Cells; Protein Kinase Inhibitors; Pyridines; src-Family Kinases; Structure-Activity Relationship; Thiazoles