Molecular and functional mapping of Plekhm1-Rab7 interaction in osteoclasts.

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases.

Single-cell dissection of Merkel cell carcinoma heterogeneity unveils transcriptomic plasticity and therapeutic vulnerabilities.

Merkel cell carcinoma (MCC), a rare but aggressive skin cancer, remains a challenge in the era of precision medicine. Immune checkpoint inhibitors (ICIs), the only approved therapy for advanced MCC, are impeded by high primary and acquired resistance. Hence, we dissect transcriptomic heterogeneity at single-cell resolution in a panel of patient tumors, revealing phenotypic plasticity in a subset of treatment-naive MCC. The tumor cells in a "mesenchymal-like" state are endowed with an inflamed phenotype that portends a better ICI response. This observation is also validated in the largest whole transcriptomic dataset available from MCC patient tumors. In contrast, ICI-resistant tumors predominantly express neuroepithelial markers in a well-differentiated state with "immune-cold" landscape. Importantly, a subtle shift to "mesenchymal-like" state reverts copanlisib resistance in primary MCC cells, highlighting potential strategies in patient stratification for therapeutics to harness tumor cell plasticity, augment treatment efficacy, and avert resistance.

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton.

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)-Cre and Cathepsin K (Ctsk)-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1ΔLysM) or differentiated osteoclasts (Tfr1ΔCtsk), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner.

Selective Inhibition of Aurora Kinase A by AK-01/LY3295668 Attenuates MCC Tumor Growth by Inducing MCC Cell Cycle Arrest and Apoptosis.

Merkel cell carcinoma (MCC) is an often-lethal skin cancer with increasing incidence and limited treatment options. Although immune checkpoint inhibitors (ICI) have become the standard of care in advanced MCC, 50% of all MCC patients are ineligible for ICIs, and amongst those treated, many patients develop resistance. There is no therapeutic alternative for these patients, highlighting the urgent clinical need for alternative therapeutic strategies. Using patient-derived genetic insights and data generated in our lab, we identified aurora kinase as a promising therapeutic target for MCC. In this study, we examined the efficacy of the recently developed and highly selective AURKA inhibitor, AK-01 (LY3295668), in six patient-derived MCC cell lines and two MCC cell-line-derived xenograft mouse models. We found that AK-01 potently suppresses MCC survival through apoptosis and cell cycle arrest, particularly in MCPyV-negative MCC cells without RB expression. Despite the challenge posed by its short in vivo durability upon discontinuation, the swift and substantial tumor suppression with low toxicity makes AK-01 a strong potential candidate for MCC management, particularly in combination with existing regimens.

The Cytoplasmic Dynein Associated Protein NDE1 Regulates Osteoclastogenesis by Modulating M-CSF and RANKL Signaling Pathways.

Cytoskeleton organization and lysosome secretion play an essential role in osteoclastogenesis and bone resorption. The cytoplasmic dynein is a molecular motor complex that regulates microtubule dynamics and transportation of cargos/organelles, including lysosomes along the microtubules. LIS1, NDE1, and NDEL1 belong to an evolutionary conserved pathway that regulates dynein functions. Disruption of the cytoplasmic dynein complex and deletion of LIS1 in osteoclast precursors arrest osteoclastogenesis. Nonetheless, the role of NDE1 and NDEL1 in osteoclast biology remains elusive. In this study, we found that knocking-down Nde1 expression by lentiviral transduction of specific shRNAs markedly inhibited osteoclastogenesis in vitro by attenuating the proliferation, survival, and differentiation of osteoclast precursor cells via suppression of signaling pathways downstream of M-CSF and RANKL as well as osteoclast differentiation transcription factor NFATc1. To dissect how NDEL1 regulates osteoclasts and bone homeostasis, we generated Ndel1 conditional knockout mice in myeloid osteoclast precursors (Ndel1ΔlysM) by crossing Ndel1-floxed mice with LysM-Cre mice on C57BL/6J background. The Ndel1ΔlysM mice developed normally. The µCT analysis of distal femurs and in vitro osteoclast differentiation and functional assays in cultures unveiled the similar bone mass in both trabecular and cortical bone compartments as well as intact osteoclastogenesis, cytoskeleton organization, and bone resorption in Ndel1ΔlysM mice and cultures. Therefore, our results reveal a novel role of NDE1 in regulation of osteoclastogenesis and demonstrate that NDEL1 is dispensable for osteoclast differentiation and function.