Inhibition of the Phosphatidylinositol-3 Kinase Pathway Using Bimiralisib in Loss-of-Function NOTCH1-Mutant Head and Neck Cancer.

BACKGROUND: PI3K/mTOR inhibition leads to apoptosis of NOTCH1-mutant head and neck squamous cell carcinoma (HNSCC) cells. We tested the efficacy of the PI3K/mTOR inhibitor bimiralisib in patients with NOTCH1-mutant HNSCC. METHODS: Patients with recurrent/metastatic NOTCH1-mutant HNSCC who had progressed during chemotherapy and immunotherapy received bimiralisib until unacceptable toxicity or progression. To assess whether NOTCH1 mutations can be detected in blood, we measured circulating tumor DNA (ctDNA). To assess activated NOTCH1 protein levels, we quantitated cleaved NOTCH1 (cl-NOTCH) by immunohistochemistry. RESULTS: Eight patients were treated, and 6 were evaluable for response. The objective response rate was 17%. For all 8 patients, median progression-free and overall survival was 5 and 7 months, respectively. Bimiralisib was well tolerated, with expected hyperglycemia. Pharmacokinetic values were consistent with published studies. NOTCH1 mutations were detected in 83.3% of ctDNA. Staining for tumor cl-NOTCH1 was negative. The trial closed early due to sponsor insolvency. CONCLUSION: Although the trial was small, outcomes with bimiralisib were better than the historical standard of care; Results will need to be confirmed in a larger trial. The lack of cl-NOTCH1 was consistent with loss-of-function mutations and validated our mutation function algorithm. The ability to detect NOTCH1 mutations in blood will help future studies. (ClinicalTrials.gov Identifier: NCT03740100).

Differential NDR/LATS interactions with the human MOB family reveal a negative role for human MOB2 in the regulation of human NDR kinases.

MOB proteins are integral components of signaling pathways controlling important cellular processes, such as mitotic exit, centrosome duplication, apoptosis, and cell proliferation in eukaryotes. The human MOB protein family consists of six distinct members (human MOB1A [hMOB1A], -1B, -2, -3A, -3B, and -3C), with hMOB1A/B the best studied due to their putative tumor-suppressive functions through the regulation of NDR/LATS kinases. The roles of the other MOB proteins are less well defined. Accordingly, we characterized all six human MOB proteins in the context of NDR/LATS binding and their abilities to activate NDR/LATS kinases. hMOB3A/B/C proteins neither bind nor activate any of the four human NDR/LATS kinases. We found that both hMOB2 and hMOB1A bound to the N-terminal region of NDR1. However, our data suggest that the binding modes differ significantly. Our work revealed that hMOB2 competes with hMOB1A for NDR binding. hMOB2, in contrast to hMOB1A/B, is bound to unphosphorylated NDR. Moreover, RNA interference (RNAi) depletion of hMOB2 resulted in increased NDR kinase activity. Consistent with these findings, hMOB2 overexpression interfered with the functional roles of NDR in death receptor signaling and centrosome overduplication. In summary, our data indicate that hMOB2 is a negative regulator of human NDR kinases in biochemical and biological settings.

Ablation of the kinase NDR1 predisposes mice to the development of T cell lymphoma.

Defective apoptosis contributes to the development of various human malignancies. The kinases nuclear Dbf2-related 1 (NDR1) and NDR2 mediate apoptosis downstream of the tumor suppressor proteins RASSF1A (Ras association domain family member 1A) and MST1 (mammalian Ste20-like kinase 1). To further analyze the role of NDR1 in apoptosis, we generated NDR1-deficient mice. Although NDR1 is activated by both intrinsic and extrinsic proapoptotic stimuli, which indicates a role for NDR1 in regulating apoptosis, NDR1-deficient T cells underwent apoptosis in a manner similar to that of wild-type cells in response to different proapoptotic stimuli. Analysis of the abundances of NDR1 and NDR2 proteins revealed that loss of NDR1 was functionally compensated for by an increase in the abundance of NDR2 protein. Despite this compensation, NDR1(-/-) and NDR1(+/-) mice were more prone to the development of T cell lymphomas than were wild-type mice. Tumor development in mice and humans was accompanied by a decrease in the overall amounts of NDR proteins in T cell lymphoma samples. Thus, reduction in the abundance of NDR1 triggered a decrease in the total amount of both isoforms. Together, our data suggest that a reduction in the abundances of the NDR proteins results in defective responses to proapoptotic stimuli, thereby facilitating the development of tumors.

The MST1 and hMOB1 tumor suppressors control human centrosome duplication by regulating NDR kinase phosphorylation.

BACKGROUND: Human MST/hSAV/LATS/hMOB tumor suppressor cascades are regulators of cell death and proliferation; however, little is known about other functions of MST/hMOB signaling. Mob1p, one of two MOB proteins in yeast, appears to play a role in spindle pole body duplication (the equivalent of mammalian centrosome duplication). We therefore investigated the role of human MOB proteins in centrosome duplication. We also addressed the regulation of human centrosome duplication by mammalian serine/threonine Ste20-like (MST) kinases, considering that MOB proteins can function together with Ste20-like kinases in eukaryotes. RESULTS: By studying the six human MOB proteins and five MST kinases, we found that MST1/hMOB1 signaling controls centrosome duplication. Overexpression of hMOB1 caused centrosome overduplication, whereas RNAi depletion of hMOB1 or MST1 impaired centriole duplication. Significantly, we delineated an hMOB1/MST1/NDR1 signaling pathway regulating centrosome duplication. More specifically, analysis of shRNA-resistant hMOB1 and NDR1 mutants revealed that a functional NDR/hMOB1 complex is critical for MST1 to phosphorylate NDR on the hydrophobic motif that in turn is required for human centrosome duplication. Furthermore, shRNA-resistant MST1 variants revealed that MST1 kinase activity is crucial for centrosome duplication whereas MST1 binding to the hSAV and RASSF1A tumor suppressor proteins is dispensable. Finally, by studying the PLK4/HsSAS-6/CP110 centriole assembly machinery, we also observed that normal daughter centriole formation depends on intact MST1/hMOB1/NDR signaling, although HsSAS-6 centriolar localization is not affected. CONCLUSIONS: Our observations propose a novel pathway in control of human centriole duplication after recruitment of HsSAS-6 to centrioles.

NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis.

Human NDR1 and 2 (NDR1/2) are serine-threonine protein kinases in a subgroup of the AGC kinase family. The mechanisms of physiological NDR1/2 activation and their function remain largely unknown. Here we report that Fas and TNF-alpha receptor stimulation activates human NDR1/2 by promoting phosphorylation at the hydrophobic motif (Thr444/442). Moreover, NDR1/2 are essential for Fas receptor-induced apoptosis as shown by the fact that NDR knockdown significantly reduced cell death whereas overexpression of the NDR1 kinase further potentiated apoptosis. Activation of NDR1/2 by death receptor stimulation is mediated by the tumor suppressor RASSF1A. Furthermore, RASSF1A-induced apoptosis largely depends on the presence of NDR1/2. Fas receptor stimulation promoted direct phosphorylation and activation of NDR1/2 by the mammalian STE20-like kinase 1 (MST1), a downstream effector of RASSF1A. Concurrently, the NDR1/2 coactivator MOB1 induced MST1-NDR-MOB1 complex formation, which is crucial for MST1-induced NDR1/2 phosphorylation upon induction of apoptosis. Our findings identify NDR1/2 as novel proapoptotic kinases and key members of the RASSF1A/MST1 signaling cascade.

The human tumour suppressor LATS1 is activated by human MOB1 at the membrane.

Downregulation of the LATS1 tumour suppressor protein kinase contributes to tumour formation in mammals and flies. Strikingly, the tumour suppressor activity depends on the interaction with Dmob (Drosphila Mps1-One binder) in Drosophila melanogaster. Recently, human LATS1 was reported to interact with human MOB1 (hMOB1), but the activation of LATS1 was not addressed. Here, we identified a highly conserved hMOB1-binding motif within LATS1's primary structure. While co-expression of LATS1 with hMOB1 did not elevate LATS1 kinase activity in mammalian cells, membrane-targeting of hMOB1 resulted in a significant increase of LATS1 activity. This stimulation was dependent on intact activation segment and hydrophobic motif phosphorylation sites, and was further found to occur a few minutes after membrane association. Therefore, we suggest a potential in vivo mechanism of LATS1 activation through rapid recruitment to the plasma membrane by hMOB1 followed by multi-site phosphorylation, thereby providing insight into the molecular regulation of the LATS tumour suppressor.