Potential Candidate Genes for Therapeutic Targeting in Chronic Myeloid Leukemia: A Pilot Study.

BACKGROUND: Chronic myeloid leukemia (CML) is a prevalent hematological malignancy known for the presence of the Philadelphia chromosome and activation of the BCR-Abl kinase activity. Although tyrosine kinase inhibitors are widely used as the standard treatment, resistance remains a concern among certain patients. This study aimed to investigate the gene expression profile of a group of CML patients in comparison to a control group in order to identify novel candidate genes associated with the disease. METHODS: Whole transcriptome sequencing was performed, and gene expression levels were validated using quantitative real-time PCR. Additionally, single nucleotide and insertion/deletion variants were analyzed in the selected candidate genes among 10 CML patients and 4 healthy control subjects. RESULTS: Analysis revealed a set of differentially expressed genes, whose up- or downregulation was further confirmed by qRT-PCR. Among the upregulated genes in the patient group were ribosomal protein like (RPL) members, specifically RPL9, RPL34, RPL36A, and RPL39, while downregulation was observed in CCDC170, LDB1, and SBF1 compared to the healthy subjects. Furthermore, gene variant studies identified novel genetic changes in these candidate genes, suggesting potential clinical significance in CML. CONCLUSIONS: This study highlights RPL9, RPL34, RPL36A, RPL39, CCDC170, LDB1, and SBF1 as potential targets in CML. Additionally, it underscores the importance of investigating these genes and their variants in larger cohort studies to assess their clinical significance in CML patients.

Targeting the PTP1B-Bcr-Abl1 interaction for the degradation of T315I mutant Bcr-Abl1 in chronic myeloid leukemia.

Small-molecule-induced degradation of mutant Bcr-Abl1 provides a potential approach to overcome Bcr-Abl1 tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML). Our previous study reported that a synthetic steroidal glycoside SBF-1 showed remarkable anti-CML activity by inducing the degradation of native Bcr-Abl1 protein. Here, we observed the comparable growth inhibition for SBF-1 in CML cells harboring T315I mutant Bcr-Abl1 in vitro and in vivo. SBF-1 triggered its degradation through disrupting the interaction between protein-tyrosine phosphatase 1B (PTP1B) and Bcr-Abl1. Using SBF-1 as a tool, we found that Tyr46 in the PTP1B catalytic domain and Tyr852 in the Bcr-Abl1 pleckstrin-homology (PH) domain are critical for their interaction. Moreover, the phosphorylation of Tyr1086 within the Bcr-Abl1 SH2 domain recruited the E3 ubiquitin ligase c-Cbl to catalyze K27-linked ubiquitin chains, which serve as a recognition signal for p62-dependent autophagic degradation. PTP1B dephosphorylated Bcr-Abl1 at Tyr1086 and prevented the recruitment of c-Cbl, leading to the stability of Bcr-Abl1. This study unravels the action mechanism of PTP1B in stabilizing Bcr-Abl1 protein and indicates that the PTP1B-Bcr-Abl1 interaction might be one of druggable targets for TKI-resistant CML with point mutations.

Targeted massively parallel sequencing of candidate regions on chromosome 22q predisposing to multiple schwannomas: An analysis of 51 individuals in a single-center experience.

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.

Bi-allelic variants in MTMR5/SBF1 cause Charcot-Marie-Tooth type 4B3 featuring mitochondrial dysfunction.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) type 4B3 (CMT4B3) is a rare form of genetic neuropathy associated with variants in the MTMR5/SBF1 gene. MTMR5/SBF1 is a pseudophosphatase predicted to regulate endo-lysosomal trafficking in tandem with other MTMRs. Although almost ubiquitously expressed, pathogenic variants primarily impact on the peripheral nervous system, corroborating the involvement of MTMR5/SBF1 and its molecular partners in Schwann cells-mediated myelinization. CASE PRESENTATION: We report a case of severe CMT4B3 characterized by early-onset motor and axonal polyneuropathy in an Italian child in absence of any evidence of brain and spine MRI abnormalities or intellectual disability and with a biochemical profile suggestive of mitochondrial disease. Using an integrated approach combining both NGS gene panels and WES analysis, we identified two novel compound heterozygous missense variants in MTMR5/SBF1 gene, p.R763H (c.2291G > A) and p.G1064E (c.3194G > A). Studies in muscle identified partial defects of oxidative metabolism. CONCLUSION: We describe the first case of an early onset severe polyneuropathy with motor and axonal involvement, due to recessive variants in the MTMR5/SBF1 gene, with no evidence of brain and spine MRI abnormalities, intellectual disability, no clinical and neurophysiological evidences of distal sensory impairment, and rapid neuromuscular deterioration. This report suggests that MTMR5/SBF1 should be considered in cases of infantile-onset CMT with secondary mitochondrial dysfunction.

Selective targeting of the androgen receptor-DNA binding domain by the novel antiandrogen SBF-1 and inhibition of the growth of prostate cancer cells.

Prostate cancers are reliant on androgens for growth and survival. Clinicians and researchers are looking for potent treatments for the resistant forms of prostate cancer; however, a handful of small molecules used in the treatment of castration-resistant prostate cancer have not shown potent effects owing to the mutations in the AR (Androgen Receptor). We used SBF-1, a well-characterized antitumor agent with potent cytotoxic effects against different kinds of cancers and investigated its effect on human prostate cancer. SBF-1 substantially inhibited the proliferation, induced apoptosis, and caused cell cycle arrest in LNCaP and PC3/AR+ prostate cancer cell lines. SBF-1 inhibited the activation of the IGF-1-PNCA pathway, as demonstrated by decreased expression of IGF-1 (insulin-like growth factor 1), proliferating cell nuclear antigen (PCNA), and its downstream Bcl-2 protein. Using microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) assays, we observed a direct binding of SBF-1 to the AR. SBF-1 binds to the AR-DBD (DNA-binding domain) and blocks the transcription of its target gene. SBF-1 demonstrated a potent antitumor effect in vivo; it inhibited AR signaling and suppressed tumor growth in animals. Our study suggests that SBF-1 is an inhibitor of the AR and might be used in the treatment of prostate cancer.

A novel frameshift deletion in autosomal recessive SBF1-related syndromic neuropathy with necklace fibres.

OBJECTIVE: To identify the genetic cause of complex neuropathy in two siblings from a consanguineous family. METHODS: The patients were recruited from our clinic. Muscle biopsy and whole-exome sequencing (WES) were performed. Fibroblasts cell lines from the index patient, unaffected parents, and three normal controls were used for cDNA analysis and western blot. RESULTS: The index patient was a 29-year-old male with clinical phenotype of syndactyly, pes cavus, swallowing difficulties, vision problem, imbalance, and muscle weakness. The sibling had similar, but milder symptoms. Nerve conduction studies and electromyography of both patients suggested sensory-motor axonal neuropathy. Muscle biopsy showed a feature of necklace fibres. WES identified a novel homozygous frameshift deletion (c.5477-5478del; p.1826-1826del) in exon 40 of the SBF1 gene in the two siblings, while both parents and the unaffected sibling were heterozygous carriers. Functional analysis showed a markedly reduced level of MTMR5 protein encoded by SBF1 in the index case. The levels of MTMR5 protein in unaffected parents were similar to those found in controls. CONCLUSION: A novel homozygous frameshift deletion in SBF1 was identified in this family. Sensory-motor axonal neuropathy and necklace fibres in biopsy were the major features expanding the phenotypic spectrum of SBF1-related recessive syndromic neuropathy.

SBF-1 inhibits contact hypersensitivity in mice through down-regulation of T-cell-mediated responses.

BACKGROUND: T lymphocytes play an important role in contact hypersensitivity. This study aims to explore the immunosuppressive activity of SBF-1, an analog of saponin OSW-1, against T lymphocytes in vitro and in vivo. METHODS: Proliferation of T lymphocytes from lymph nodes of mice was determined by MTT assay. Flow cytometry analysis was performed to assess T cell activation and apoptosis. Levels of cytokines were determined by PCR and ELISA. BALB/c mice were sensitized and challenged with picryl chloride and thickness of left and right ears were measured. RESULTS: SBF-1 effectively inhibited T lymphocytes proliferation induced by concanavalin A (Con A) or anti-CD3 plus anti-CD28 at a very low dose (10 nM) but exhibited little toxicity in non-activated T lymphocytes at concentrations up to 10 µM. In addition, SBF-1 inhibited the expression of CD25 and CD69, as well as he phosphorylation of AKT in Con A-activated T cells. SBF-1 also induced apoptosis of activated T cells. In addition, SBF-1 also downregulated the induction of the T cell cytokines, IL-2 and IFN-γ in a dose-dependent manner. Furthermore, SBF-1 significantly suppressed ear swelling and inflammation in a mouse model of picryl chloride-induced contact hypersensitivity. CONCLUSIONS: Our findings suggest that SBF-1 has an unique immunosuppressive activity both in vitro and in vivo mainly through inhibiting T cell proliferation and activation. Its mechanism appears to be related to the blockage of AKT signaling pathway.

Discovery of SBF1 as an allosteric inhibitor targeting the PIF-pocket of 3-phosphoinositide-dependent protein kinase-1.

3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a crucial role in the signal transduction of massive growth-related protein kinases. In this work, a computational study has been performed to investigate the binding pose of the hydrolyzed product of SBF1 (SBF1-) with PDK1. The binding pose was predicted by Vina and was further refined in a molecular dynamics simulation. For comparison, four published low molecular weight compounds (PS48, PS171, PS182, and PS210) binding with PDK1 were also studied. SBF1- was anchored in the PIF-pocket of PDK1 with salt bridge interaction using its carboxylate moiety, which is a common feature among the known ligands. Hydrogen bonds to THR148 and vdW interactions with GLN150 also have contributions to the association affinity. The allosteric regulation on PDK1 via the binding of SBF1- was further addressed. The binding affinity of SBF1- in complex with PDK1 is comparable to those of PS171 and PS182, with an estimated IC50 in a range from 2.0 to 10.0 µ molar. Comparison between the free energy profiles with the presence or absence of SBF1- in the binding pocket indicates that the binding of SBF1- enhances the hinge motion and suppresses the fluctuation of the end-to-end distance in α B of PDK1. These results demonstrate that SBF1- is a promising allosteric regulator of PDK1 targeting the PIF-binding pocket and can serve as a new scaffold template for the design of new drugs targeting PDK1.

SBF-1 preferentially inhibits growth of highly malignant human liposarcoma cells.

Frequent local recurrence and metastasis are generally involved in human liposarcoma, but the management is a challenge. There is an urgent need for improved effective therapy. In the present study, we reported that SBF-1, a steroidal glycoside, inhibited the growth of cultured highly malignant human liposarcoma SW872-S cells in vitro and in vivo. SBF-1 down-regulated the phosphorylation of protein kinase B (AKT) and thus reduced cell adhesion to fibronectin and laminin. Then we found that SBF-1 inhibited the expression of oxysterol binding protein (OSBP) in SW872-S cells, indicating that OSBP may be involved in malignant liposarcoma cell survival. Cancer cell growth and AKT phosphorylation were inhibited significantly upon knockdown of OSBP in SW872-S cells in vitro. Taken together, these results suggest that SBF-1 causes an apparent loss of OSBP function in SW872-S cells, resulting in growth inhibition. Based on our findings, OSBP serves as a potential therapeutic target for human liposarcoma.

Novel SBF1 splice-site null mutation broadens the clinical spectrum of Charcot-Marie-Tooth type 4B3 disease.

Four siblings of consanguineous Bedouin kindred presented at infancy with an autosomal recessive syndrome of congenital microcephaly, facial dysmorphism, strabismus, developmental delay and ataxia with positive pyramidal signs. Toward the end of their first decade, they developed areflexia, multiple cranial neuropathies and severe polyneuropathy with progressive muscle weakness, affecting proximal and distal extremities. Physical assessment exhibited kyphoscoliosis, bilateral syndactyly and distal muscle wasting with drop-foot and pes cavus. Magnetic resonance imaging (MRI) showed profound cerebellar atrophy with highly unique findings at the pontine and mesencephalic levels, previously described as "fork and bracket" signs. Genome-wide linkage analysis identified a single ~1.5 Mbp disease-associated locus on chromosome 22q13.33. Whole exome sequencing identified a single novel homozygous deleterious splice-site mutation within this locus in SET binding factor 1 (SBF1). SBF1 missense mutations were shown to underlie Charcot-Marie-Tooth (CMT) type 4B3 disease, a rare autosomal recessive subtype of CMT4. The novel SBF1 null mutation highlights distinct severe phenotypic manifestations, broadening the clinical spectrum of SBF1-related neuropathies: cerebellar and pyramidal signs evident in the first months of life with peripheral polyneuropathy emerging only toward the end of the first decade, together with unique MRI findings.

WANTED - Dead or alive: Myotubularins, a large disease-associated protein family.

Myotubularins define a large family of proteins conserved through evolution. Several members are mutated in different neuromuscular diseases including centronuclear myopathies and Charcot-Marie-Tooth (CMT) neuropathies, or are linked to a predisposition to obesity and cancer. While some members have phosphatase activity against the 3-phosphate of phosphoinositides, regulating the phosphorylation status of PtdIns3P and PtdIns(3,5)P2 implicated in membrane trafficking and autophagy, and producing PtdIns5P, others lack key residues in the catalytic site and are classified as dead-phosphatases. However, these dead phosphatases regulate phosphoinositide-dependent cellular pathways by binding to catalytically active myotubularins. Here we review previous studies on the molecular regulation and physiological roles of myotubularins. We also used the recent myotubularins three-dimensional structures to underline key residues that are mutated in neuromuscular diseases and required for enzymatic activity. In addition, through database mining and analysis, expression profile and specific isoforms of the different myotubularins are described in depth, as well as a revisited protein interaction network. Comparison of the interactome and expression data for each myotubularin highlights specific protein complexes and tissues where myotubularins should have a key regulatory role.

SBF1 mutations associated with autosomal recessive axonal neuropathy with cranial nerve involvement.

Biallelic mutations in the SBF1 gene have been identified in one family with demyelinating Charcot-Marie-Tooth disease (CMT4B3) and two families with axonal neuropathy and additional neurological and skeletal features. Here we describe novel sequence variants in SBF1 (c.1168C>G and c.2209_2210del) as the potential causative mutations in two siblings with severe axonal neuropathy, hearing loss, facial weakness and bulbar features. Pathogenicity of these variants is supported by co-segregation and in silico analyses and evolutionary conservation. Our findings suggest that SBF1 mutations may cause a syndromic form of autosomal recessive axonal neuropathy (AR-CMT2) in addition to CMT4B3.

Blockade of the interaction between Bcr-Abl and PTB1B by small molecule SBF-1 to overcome imatinib-resistance of chronic myeloid leukemia cells.

In this study, a synthetic steroidal glycoside SBF-1 had strong and preferential antitumor effects on the human chronic myeloid leukemia (CML) cell line K562 and its imatinib-resistant form K562/G. SBF-1 induced apoptosis in both cell lines without any effect on cell cycle arrest. It also inhibited the activation of PI3K/Akt pathway members, such as PI3K and Akt, as well as downstream targets mTOR and Bcl-2. Moreover, the degradation of the Bcr-Abl protein was induced by SBF-1 in a concentration- and time-dependent manner. Using a pull-down assay, SBF-1 was found to bind to both Bcr-Abl and PTP1B and disrupted the interaction between them. SBF-1 triggered the degradation of Bcr-Abl through ubiquitination via the lysosome pathway. Taking together these findings, this study, for the first time, suggests that the blockade of the interaction between Bcr-Abl and PTP1B may be a feasible strategy for the treatment of CML, especially CML with resistance to Bcr-Abl kinase inhibitor imatinib. Our study also indicates that SBF-1 may serve as a leading compound for novel anti-CML therapeutic agents.

Genome-Engineering Tools to Establish Accurate Reporter Cell Lines That Enable Identification of Therapeutic Strategies to Treat Friedreich's Ataxia.

Friedreich's ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genome-engineering tools that enable the generation of human cell lines that express the frataxin gene fused to a luciferase reporter gene from its endogenous locus. Performing a pilot high-throughput genomic screen in a newly established reporter cell line, we uncovered novel negative regulators of frataxin expression. Rational design of small-molecule inhibitors of the identified frataxin repressors and/or high-throughput screening of large siRNA or compound libraries with our system may yield treatments for Friedreich's ataxia.

DNA microarray reveals ZNF195 and SBF1 are potential biomarkers for gemcitabine sensitivity in head and neck squamous cell carcinoma cell lines.

Gemcitabine is a potential chemotherapy drug for treatment of head and neck squamous cell carcinoma (HNSCC), however, the poor or partial response of HNSCC patients to gemcitabine demonstrated the urgent need for gemcitabine biomarkers to improve the therapy. In present work, 10 HNSCC cell lines were employed to figure out the biomarkers for gemcitabine sensitivity. The sensitivities of these 10 cell lines to gemcitabine and the basal expression of these cell lines was investigated, the correlation between gemcitabine response (IC50 dose) and gene expression was investigated by Pearson correlation and FDR estimation. The top seven positive genes responsible for gemcitabine sensitivity were validated by qPCR in these 10 HNSCC cell lines, while only two genes (SBF1 and ZNF195) were expression-correlated to gemcitabine response. Furthermore, ZNF195 expression was closely associated with gemcitabine sensitivity in the subsequent independent validation in cell lines from various types of cancer. Our work might provide potential biomarkers for gemcitabine sensitivity in HNSCC and various type of cancer.