Exploiting the fibroblast growth factor receptor-1 vulnerability to therapeutically restrict the MYC-EZH2-CDKN1C axis-driven proliferation in Mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a lethal hematological malignancy with a median survival of 4 years. Its lethality is mainly attributed to a limited understanding of clinical tumor progression and resistance to current therapeutic regimes. Intrinsic, prolonged drug treatment and tumor-microenvironment (TME) facilitated factors impart pro-tumorigenic and drug-insensitivity properties to MCL cells. Hence, elucidating neoteric pharmacotherapeutic molecular targets involved in MCL progression utilizing a global "unified" analysis for improved disease prevention is an earnest need. Using integrated transcriptomic analyses in MCL patients, we identified a Fibroblast Growth Factor Receptor-1 (FGFR1), and analyses of MCL patient samples showed that high FGFR1 expression was associated with shorter overall survival in MCL patient cohorts. Functional studies using pharmacological intervention and loss of function identify a novel MYC-EZH2-CDKN1C axis-driven proliferation in MCL. Further, pharmacological targeting with erdafitinib, a selective small molecule targeting FGFRs, induced cell-cycle arrest and cell death in-vitro, inhibited tumor progression, and improved overall survival in-vivo. We performed extensive pre-clinical assessments in multiple in-vivo model systems to confirm the therapeutic potential of erdafitinib in MCL and demonstrated FGFR1 as a viable therapeutic target in MCL.

Evolution of m6A-related genes in insects and the function of METTL3 in silkworm embryonic development.

N6-methyladenosine (m6A) plays a key role in many biological processes. However, the function and evolutionary relationship of m6A-related genes in insects remain largely unknown. Here we analysed the phylogeny of m6A-related genes among 207 insect species and found that m6A-related genes are evolutionarily conserved in insects. Subcellular localization experiments of m6A-related proteins in BmN cells confirmed that BmYTHDF3 was localized in the cytoplasm, BmMETTL3, BmMETTL14, and BmYTHDC were localized in the nucleus, and FL2D was localized to both the nucleus and cytoplasm. We examined the expression patterns of m6A-related genes during the embryonic development of Bombyx mori. To elucidate the function of BmMETTL3 during the embryonic stage, RNA sequencing was performed to measure changes in gene expression in silkworm eggs after BmMETTL3 knockdown, as well as in BmN cells overexpressing BmMETTL3. The global transcriptional pattern showed that knockdown of BmMETTL3 affected multiple cellular processes, including oxidoreductase activity, transcription regulator activity, and the cation binding. In addition, transcriptomic data revealed that many observed DEGs were associated with fundamental metabolic processes, including carbon metabolism, purine metabolism, amino acid biosynthesis, and the citrate cycle. Interestingly, we found that knockdown of BmMETTL3 significantly affected Wnt and Toll/Imd pathways in embryos. Taken together, these results suggest that BmMETTL3 plays an essential role in the embryonic development of B. mori, and deepen our understanding of the function of m6A-related genes in insects.

PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Response and resistance to CDK12 inhibition in aggressive B-cell lymphomas.

Despite significant progress in the treatment of patients with diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), the prognosis of patients with relapsed disease remains poor due to the emergence of drug resistance and subsequent disease progression. Identification of novel targets and therapeutic strategies for these diseases represents an urgent need. Here, we report that both MCL and DLBCL are exquisitely sensitive to transcription-targeting drugs, in particular THZ531, a covalent inhibitor of cyclin-dependent kinase 12 (CDK12). By implementing pharmacogenomics and a cell-based drug screen, we found that THZ531 leads to inhibition of oncogenic transcriptional programs, especially the DNA damage response pathway, MYC target genes and the mTOR-4EBP1-MCL-1 axis, contributing to dramatic lymphoma suppression in vitro. We also identified de novo and established acquired THZ531-resistant lymphoma cells conferred by over-activation of the MEK-ERK and PI3K-AKT-mTOR pathways and upregulation of multidrug resistance-1 (MDR1) protein. Of note, EZH2 inhibitors reversed resistance to THZ531 by competitive inhibition of MDR1 and, in combination with THZ531, synergistically inhibited MCL and DLBCL growth in vitro. Our study indicates that CDK12 inhibitors, alone or together with EZH2 inhibitors, offer promise as novel effective approaches for difficult-to-treat DLBCL and MCL.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

HDAC11 regulates expression of C/EBPß and immunosuppressive molecules in myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) constitute a heterogeneous population of immature myeloid cells derived from bone marrow and negatively regulate both innate and adaptive immunity in the tumor microenvironment. Previously we have demonstrated that MDSCs lacking histone deacetylase 11 (HDAC11) displayed an increased suppressive activity against CD8+ T-cells. However, the mechanisms of HDAC11 that contribute to the suppressive function of MDSCs remain unclear. Here, we show that arginase activity and NO production is significantly higher in HDAC11 knockout MDSCs when compared with wild-type (WT) controls. In the absence of HDAC11, elevated arginase level and enzymatic activity were observed preferentially in the tumor-infiltrated granulocytic MDSCs, whereas iNOS expression and NO production were increased in the tumor-infiltrated monocytic MDSCs. Of note and for the first time, we demonstrated an association between the elevated expression of immunosuppressive molecules with up-regulation of the transcription factor C/EBPß in MDSCs lacking HDAC11. Interestingly, the highest expression of C/EBPß was observed among CD11b+ Gr-1+ MDSCs isolated from tumor-bearing mice. The additional demonstration that HDAC11 is recruited to the promoter region of C/EBPß in WT MDSCs suggests a novel molecular mechanism by which HDAC11 influence the expression of immunosuppressive molecules in MDSCs through regulation of C/EBPß gene expression.

Transcriptional programming drives Ibrutinib-resistance evolution in mantle cell lymphoma.

Ibrutinib, a bruton's tyrosine kinase (BTK) inhibitor, provokes robust clinical responses in aggressive mantle cell lymphoma (MCL), yet many patients relapse with lethal Ibrutinib-resistant (IR) disease. Here, using genomic, chemical proteomic, and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally, and importantly, we find that a robust and facile ex vivo image-based functional drug screening platform can predict clinical therapeutic responses of IR MCL and identify vulnerabilities that can be targeted to disable the evolution of IR.

Bisphosphonates for delivering drugs to bone.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Targeting anti-cancer agents to bone using bisphosphonates.

The skeleton is affected by numerous primary and metastatic solid and hematopoietic malignant tumors, which can cause localized sites of osteolysis or osteosclerosis that can weaken bones and increase the risk of fractures in affected patients. Chemotherapeutic drugs can eliminate some tumors in bones or reduce their volume and skeletal-related events, but adverse effects on non-target organs can significantly limit the amount of drug that can be administered to patients. In these circumstances, it may be impossible to deliver therapeutic drug concentrations to tumor sites in bones. One attractive mechanism to approach this challenge is to conjugate drugs to bisphosphonates, which can target them to bone where they can be released at diseased sites. Multiple attempts have been made to do this since the 1990s with limited degrees of success. Here, we review the results of pre-clinical and clinical studies made to target FDA-approved drugs and other antineoplastic small molecules to bone to treat diseases affecting the skeleton, including osteoporosis, metastatic bone disease, multiple myeloma and osteosarcoma. Results to date are encouraging and indicate that drug efficacy can be increased and side effects reduced using these approaches. Despite these successes, challenges remain: no drugs have gone beyond small phase 2 clinical trials, and major pharmaceutical companies have shown little interest in the approach to repurpose any of their drugs or to embrace the technology. Nevertheless, interest shown by smaller biotechnology companies in the technology suggests that bone-targeting of drugs with bisphosphonates has a viable future.

Targeting Bortezomib to Bone Increases Its Bone Anabolic Activity and Reduces Systemic Adverse Effects in Mice.

Bortezomib (Btz) is a proteasome inhibitor approved by the FDA to treat multiple myeloma. It also increases bone volume by promoting osteoblast differentiation and inhibiting osteoclastogenesis in mice. However, Btz has severe systemic adverse effects, which would limit its use as a bone anabolic agent. Here, we designed and synthesized a bone-targeted form of Btz by conjugating it to a bisphosphonate (BP) with no antiresorptive activity. We report that BP-Btz inhibited osteoclast formation and bone resorption and stimulated osteoblast differentiation in vitro similar to Btz. In vivo, BP-Btz increased bone volume more effectively than Btz in three mouse models: untreated wild-type mice, mice with ovariectomy, and aged mice with tibial factures. Importantly, BP-Btz had significantly less systemic side effects than Btz, including less thymic cell death, sympathetic nerve damage, and thrombocytopenia, and it improved survival rates in aged mice. Thus, BP-Btz represents a novel anabolic agent to treat conditions, such as postmenopausal and age-related bone loss. Bone targeting is an attractive approach to repurpose approved drugs to treat skeletal diseases. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models.

Drug-tolerant "persister" tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.

Synthesis of a Bone-Targeted Bortezomib with In Vivo Anti-Myeloma Effects in Mice.

Multiple myeloma (MM) is the most common cancer affecting the bone and bone marrow and remains incurable for most patients; novel therapies are therefore needed. Bortezomib (Btz) is an FDA-approved drug for the treatment of patients with MM. However, its severe side effects require a dose reduction or the potential discontinuation of treatment. To overcome this limitation, we conjugated Btz to a bisphosphonate (BP) residue lacking anti-osteoclastic activity using a novel chemical linker and generated a new bone-targeted Btz-based (BP-Btz) proteasome inhibitor. We demonstrated that BP-Btz, but not Btz, bound to bone slices and inhibited the growth of MM cells in vitro. In a mouse model of MM, BP-Btz more effectively reduced tumor burden and bone loss with less systemic side effects than Btz. Thus, BP-Btz may represent a novel therapeutic approach to treat patients with MM.

PLK1 stabilizes a MYC-dependent kinase network in aggressive B cell lymphomas.

Concordant activation of MYC and BCL-2 oncoproteins in double-hit lymphoma (DHL) results in aggressive disease that is refractory to treatment. By integrating activity-based proteomic profiling and drug screens, polo-like kinase-1 (PLK1) was identified as an essential regulator of the MYC-dependent kinome in DHL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression, and connoted poor outcome. Further, PLK1 signaling augmented MYC protein stability, and in turn, MYC directly induced PLK1 transcription, establishing a feed-forward MYC-PLK1 circuit in DHL. Finally, inhibition of PLK1 triggered degradation of MYC and of the antiapoptotic protein MCL-1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL cell growth, survival, and tumorigenicity, supporting clinical targeting of PLK1 in DHL.