An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation.

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.

CBFß-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia.

The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

Isocitrate Dehydrogenase Mutation and (R)-2-Hydroxyglutarate: From Basic Discovery to Therapeutics Development.

The identification of heterozygous mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) in subsets of cancers, including secondary glioblastoma, acute myeloid leukemia, intrahepatic cholangiocarcinoma, and chondrosarcomas, led to intense discovery efforts to delineate the mutations' involvement in carcinogenesis and to develop therapeutics, which we review here. The three IDH isoforms (nicotinamide adenine dinucleotide phosphate-dependent IDH1 and IDH2, and nicotinamide adenine dinucleotide-dependent IDH3) contribute to regulating the circuitry of central metabolism. Several biochemical and genetic observations led to the discovery of the neomorphic production of the oncometabolite (R)-2-hydroxyglutarate (2-HG) by mutant IDH1 and IDH2 (mIDH). Heterozygous mutation of IDH1/2 and accumulation of 2-HG cause profound metabolic and epigenetic dysregulation, including inhibition of normal cellular differentiation, leading to disease. Crystallographic structural studies during the development of compounds targeting mIDH demonstrated common allosteric inhibition by distinct chemotypes. Ongoing clinical trials in patients with mIDH advanced hematologic malignancies have demonstrated compelling clinical proof-of-concept, validating the biology and drug discovery approach.

Treatment for hypoactive sexual desire.

Flibanserin acts at cortical, limbic, hypothalamic, and brainstem nuclei to inhibit serotonin release by binding to 5-HT1A autoreceptors and block postsynaptic action of serotonin at 5-HT2A receptors. This gradually disinhibits the turnover of other monoamines like dopamine and noradrenaline that are critical for sexual desire.

Fixing a hole where the Ras gets in.

A clinically efficacious Ras inhibitor has eluded drug-discovery efforts for decades. In a paper in Nature, Zimmermann and et al. show that blocking a hole in PDEδ that normally engages the lipid tail of Ras disrupts downstream signaling, pointing to a potentially promising route to develop Ras inhibitors for cancer treatment.

Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

A phase 3 randomized trial of mavorixafor, a CXCR4 antagonist, for WHIM syndrome.

ABSTRACT: We investigated efficacy and safety of mavorixafor, an oral CXCR4 antagonist, in participants with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency caused by CXCR4 gain-of-function variants. This randomized (1:1), double-blind, placebo-controlled, phase 3 trial enrolled participants aged ≥12 years with WHIM syndrome and absolute neutrophil count (ANC) ≤0.4 × 103/µL. Participants received once-daily mavorixafor or placebo for 52 weeks. The primary end point was time (hours) above ANC threshold ≥0.5 × 103/µL (TATANC; over 24 hours). Secondary end points included TAT absolute lymphocyte count ≥1.0 × 103/µL (TATALC; over 24 hours); absolute changes in white blood cell (WBC), ANC, and absolute lymphocyte count (ALC) from baseline; annualized infection rate; infection duration; and total infection score (combined infection number/severity). In 31 participants (mavorixafor, n = 14; placebo, n = 17), mavorixafor least squares (LS) mean TATANC was 15.0 hours and 2.8 hours for placebo (P < .001). Mavorixafor LS mean TATALC was 15.8 hours and 4.6 hours for placebo (P < .001). Annualized infection rates were 60% lower with mavorixafor vs placebo (LS mean 1.7 vs 4.2; nominal P = .007), and total infection scores were 40% lower (7.4 [95% confidence interval [CI], 1.6-13.2] vs 12.3 [95% CI, 7.2-17.3]). Treatment with mavorixafor reduced infection frequency, severity, duration, and antibiotic use. No discontinuations occurred due to treatment-emergent adverse events (TEAEs); no related serious TEAEs were observed. Overall, mavorixafor treatment demonstrated significant increases in LS mean TATANC and TATALC, reduced infection frequency, severity/duration, and was well tolerated. The trial was registered at www.clinicaltrials.gov as #NCT03995108.

Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel ß-tubulin benzimidazole resistance mutation.

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 ß-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 ß-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans ß-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

Candesartan upregulates angiotensin-converting enzyme 2 in kidneys of male animals by decreased ubiquitination.

Candesartan is a common angiotensin-II receptor-1 blocker used for patients with cardiovascular and renal diseases. Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of blood pressure (BP), and also a major receptor for coronaviruses. To determine whether and how candesartan upregulates ACE2, we examined BP and ACE2 in multi-organs from male and female C57BL/6J mice treated with candesartan (1 mg/kg, i.p.) for 7 days. Relative to the vehicle, candesartan lowered BP more in males than females; ACE2 protein abundances were increased in kidneys, not lungs, hearts, aorta, liver, spleen, brain, or serum, only from males. Ace2-mRNA was similar in kidneys. Candesartan also decreased BP in normal, hypertensive, and nephrotic male rats. The renal ACE2 was increased by the drug in normal and nephrotic male rats but not spontaneously hypertensive ones. In male mouse kidneys, ACE2 was distributed at sodium-hydrogen-exchanger-3 positive proximal-convoluted-tubules; ACE2-ubiquitination was decreased by candesartan, accompanied with increased ubiquitin-specific-protease-48 (USP48). In candesartan-treated mouse renal proximal-convoluted-tubule cells, ACE2 abundances and activities were increased while ACE2-ubiquitination and colocalization with lysosomal and proteosomal markers were decreased. The silence of USP48 by siRNA caused a reduction of ACE2 in the cells. Thus, the sex-differential ACE2 upregulation by candesartan in kidney from males may be due to the decreased ACE2-ubiquitination, associated with USP48, and consequent degradation in lysosomes and proteosomes. This is a novel mechanism and may shed light on candesartan-like-drug choice in men and women prone to coronavirus infections.

A molecular mechanism for angiotensin II receptor blocker-mediated slit membrane protection: Angiotensin II increases nephrin endocytosis via AT1-receptor-dependent ERK 1/2 activation.

Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.

Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks.

Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.

Distinct transcriptional profiles in rat thyroid glands after developmental exposure to three in vitro thyroperoxidase inhibiting chemicals.

Thyroperoxidase (TPO) is central in thyroid hormone (TH) synthesis and inhibition can lead to TH deficiency. Many chemicals can inhibit TPO activity in vitro, but how this may manifest in the developing thyroid gland at the molecular level is unclear. Here, we characterized the thyroid gland transcriptome of male rats developmentally exposed to the in vitro TPO-inhibitors amitrole, 2-mercaptobenzimidazole (MBI), or cyanamide by use of Bulk-RNA-Barcoding (BRB) and sequencing. Amitrole exposure caused TH deficiency and 149 differentially expressed genes in the thyroid gland. The effects indicated an activated and growing thyroid gland. MBI caused intermittent changes to serum TH concentrations in a previous study and this was accompanied by 60 differentially expressed genes in the present study. More than half of these were also affected by amitrole, indicating that they could be early effect biomarkers of developmental TH system disruption due to TPO inhibition. Further work to validate the signature is needed, including assessment of substance independency and applicability domain.

Drug Resistance Assessed in a Phase 3 Clinical Trial of Maribavir Therapy for Refractory or Resistant Cytomegalovirus Infection in Transplant Recipients.

BACKGROUND: This drug resistance analysis of a randomized trial includes 234 patients receiving maribavir and 116 receiving investigator-assigned standard therapy (IAT), where 56% and 24%, respectively, cleared cytomegalovirus DNA at week 8 (treatment responders). METHODS: Baseline and posttreatment plasma samples were tested for mutations conferring drug resistance in viral genes UL97, UL54, and UL27. RESULTS: At baseline, genotypic testing revealed resistance to ganciclovir, foscarnet, or cidofovir in 56% of patients receiving maribavir and 68% receiving IAT, including 9 newly phenotyped mutations. Among them, 63% (maribavir) and 21% (IAT) were treatment responders. Detected baseline maribavir resistance mutations were UL27 L193F (n = 1) and UL97 F342Y (n = 3). Posttreatment, emergent maribavir resistance mutations were detected in 60 (26%) of those randomized to maribavir, including 49 (48%) of 103 nonresponders and 25 (86%) of the 29 nonresponders where viral DNA initially cleared then rebounded while on maribavir. The most common maribavir resistance mutations were UL97 T409M (n = 34), H411Y (n = 26), and C480F (n = 21), first detected 26 to 130 (median 56) days after starting maribavir. CONCLUSIONS: Baseline maribavir resistance was rare. Drug resistance to standard cytomegalovirus antivirals did not preclude treatment response to maribavir. Rebound in plasma cytomegalovirus DNA while on maribavir strongly suggests emerging drug resistance. CLINICAL TRIALS REGISTRATION: NCT02931539.

Completion axillary lymph node dissection for the identification of pN2-3 status as an indication for adjuvant CDK4/6 inhibitor treatment: a post-hoc analysis of the randomised, phase 3 SENOMAC trial.

BACKGROUND: In luminal breast cancer, adjuvant CDK4/6 inhibitors (eg, abemaciclib) improve invasive disease-free survival. In patients with T1-2, grade 1-2 tumours, and one or two sentinel lymph node metastases, completion axillary lymph node dissection (cALND) is the only prognostic tool available that can reveal four or more nodal metastases (pN2-3), which is the only indication for adjuvant abemaciclib in this setting. However, this technique can lead to substantial arm morbidity in patients. We aimed to pragmatically describe the potential benefit and harm of this strategy on the individual patient level in patients from the ongoing SENOMAC trial. METHODS: In the randomised, phase 3, SENOMAC trial, patients aged 18 years or older, of any performance status, with clinically node-negative T1-T3 breast cancer and one or two sentinel node macrometastases from 67 sites in five European countries (Denmark, Germany, Greece, Italy, and Sweden) were randomly assigned (1:1), via permutated block randomisation (random block size of 2 and 4) stratified by country, to either cALND or its omission (ie, they had a sentinel lymph node biopsy only). The primary outcome is overall survival, which is yet to be reported. In this post-hoc analysis, patients from the SENOMAC per-protocol population, with luminal oestrogen-receptor positive, HER2-negative, T1-2, histological grade 1-2 breast cancer, with tumour size of 5 cm or smaller were selected to match the characteristics of cohort 1 of the monarchE trial who would only have an indication for adjuvant abemaciclib if found to have 4 or more nodal metastases. The primary study objective was to determine the number of patients who developed patient-reported severe or very severe impairment of physical arm function after cALND (as measured by the Lymphedema Functioning, Disability, and Health [Lymph-ICF] Questionnaire) 1 year after surgery to avoid one invasive disease-free survival event at 5 years with 2 years of adjuvant abemaciclib, using invasive disease-free survival event data from cohort 1 of the monarchE trial. The SENOMAC trial is registered with ClincialTrials.gov, NCT02240472, and is closed to accrual and ongoing. FINDINGS: Between Jan 31, 2015, and Dec 31, 2021, 2766 patients were enrolled in SENOMAC and randomly assigned to cALND (n=1384) or sentinel node biopsy only (n=1382), of whom 2540 were included in the per-protocol population. 1705 (67%) of 2540 patients met this post-hoc study's eligibility criteria, of whom 802 (47%) had a cALND and 903 (53%) had a sentinel lymph node biopsy only. Median age at randomisation was 62 years (IQR 52-71), 1699 (>99%) of 1705 patients were female, and six (<1%) were male. Among 1342 patients who responded to questionnaires, after a median follow-up of 45·2 months (IQR 25·6-59·8; data cutoff Nov 17, 2023), patient-reported severe or very severe impairment of physical arm function was reported in 84 (13%) of 634 patients who had cALND versus 30 (4%) of 708 who had sentinel lymph node biopsy only (χ2 test p<0·0001). To avoid one invasive disease-free survival event at 5 years with adjuvant abemaciclib, cALND would need to be performed in 104 patients, and would result in nine patients having severe or very severe impairment of physical arm function 1 year after surgery. INTERPRETATION: As a method to potentially identify an indication for abemaciclib, and subsequently avoid invasive disease-free survival events at 5 years with 2 years of adjuvant abemaciclib, cALND carries a substantial risk of severe or very severe arm morbidity and so cALND should be discouraged for this purpose. FUNDING: Swedish Research Council, the Swedish Cancer Society, the Nordic Cancer Union, and the Swedish Breast Cancer Association.

A Randomized, Double-Blind, Phase 3 Safety and Efficacy Study of Ridinilazole Versus Vancomycin for Treatment of Clostridioides difficile Infection: Clinical Outcomes With Microbiome and Metabolome Correlates of Response.

BACKGROUND: Exposure to antibiotics predisposes to dysbiosis and Clostridioides difficile infection (CDI) that can be severe, recurrent (rCDI), and life-threatening. Nonselective drugs that treat CDI and perpetuate dysbiosis are associated with rCDI, in part due to loss of microbiome-derived secondary bile acid (SBA) production. Ridinilazole is a highly selective drug designed to treat CDI and prevent rCDI. METHODS: In this phase 3 superiority trial, adults with CDI, confirmed with a stool toxin test, were randomized to receive 10 days of ridinilazole (200 mg twice daily) or vancomycin (125 mg 4 times daily). The primary endpoint was sustained clinical response (SCR), defined as clinical response and no rCDI through 30 days after end of treatment. Secondary endpoints included rCDI and change in relative abundance of SBAs. RESULTS: Ridinilazole and vancomycin achieved an SCR rate of 73% versus 70.7%, respectively, a treatment difference of 2.2% (95% CI: -4.2%, 8.6%). Ridinilazole resulted in a 53% reduction in recurrence compared with vancomycin (8.1% vs 17.3%; 95% CI: -14.1%, -4.5%; P = .0002). Subgroup analyses revealed consistent ridinilazole benefit for reduction in rCDI across subgroups. Ridinilazole preserved microbiota diversity, increased SBAs, and did not increase the resistome. Conversely, vancomycin worsened CDI-associated dysbiosis, decreased SBAs, increased Proteobacteria abundance (∼3.5-fold), and increased the resistome. CONCLUSIONS: Although ridinilazole did not meet superiority in SCR, ridinilazole greatly reduced rCDI and preserved microbiome diversity and SBAs compared with vancomycin. These findings suggest that treatment of CDI with ridinilazole results in an earlier recovery of gut microbiome health. Clinical Trials Registration.Ri-CoDIFy 1 and 2: NCT03595553 and NCT03595566.

The calcium-calmodulin-dependent protein kinase kinase inhibitor, STO-609, inhibits nicotine-induced currents and intracellular calcium increase in insect neurosecretory cells.

Insect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates and are considered to be major targets of neonicotinoid insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, in order to explore the intracellular mechanisms leading to the regulation of insect neuronal nAChRs in more detail. Using whole-cell patch-clamp and fura-2AM calcium imaging techniques, we found that a novel CaMKK/AMPK pathway could be involved in the intracellular regulation of DUM neuron nAChRs. The CaMKK selective inhibitor, STO, reduced nicotinic current amplitudes, and strongly when co-applied with α-Bgt. Interestingly, intracellular application of the AMPK activator, A-76, prevented the reduction in nicotine-induced currents observed in the presence of the AMPK inhibitor, dorsomorphin. STO prevented the increase in intracellular calcium induced by nicotine, which was not dependent on α-Bgt. Currents induced by 1 mM LMA, a selective activator of nAChR2, were reduced under bath application of STO, and mecamylamine, which blocked nAChR2 subtype, inhibited the increase in intracellular calcium induced by LMA. These findings provide insight into potential complex mechanisms linked to the modulation of the DUM neuron nAChRs and CaMKK pathway.

Association between CDK4/6 inhibitors and drug-related osteonecrosis of the jaw: A pharmacoepidemiological study using the FDA Adverse Events Reporting System.

The most common toxicities associated with cyclin-dependent kinase (CDK) 4/6 inhibitor therapy include decreased leukopenia and neutropenia due to the inhibition of CDK6 of leukocyte and neutrophil precursors in bone marrow. These hematological toxicities are more commonly observed with palbociclib administration than with abemaciclib administration, which is approximately 13 times more selective against CDK4 than CDK6. Thus, even though both successfully inhibit CDK4/6, the side effects of palbociclib and abemaciclib differ due to differences in selectivity. Recent reports have suggested an association between palbociclib and medication-related osteonecrosis of the jaw; however, reports on this association are inconsistent. This study investigated the potential association of palbociclib and abemaciclib with MRONJ using the FAERS. Signals of "Osteonecrosis of jaw" were detected only in females using palbociclib (cROR025: 2.08). Other signals detected included stomatitis-related adverse events with abemaciclib and intraoral soft tissue damage and infection with palbociclib. As previous exploratory studies have reported MRONJ signals for bisphosphonates and denosumab, we calculated the aROR for palbociclib-induced osteonecrosis of the jaw using concomitant bisphosphonates and denosumab as covariates. A signal was detected even after adjusting for sex, age, and concomitant medications as covariates (aROR0025: 5.74). A proper understanding of the differences in CDK selectivity is necessary for the appropriate use of CDK4/6 inhibitors. To the best of our knowledge, this is the first report on CDK4/6 inhibitors and drug-related osteonecrosis of the jaw. We believe that these results will offer new insights into adverse events related to the use of CDK4/6 inhibitors, and may aid in the proper use of CDK4/6 inhibitors.

Glasdegib with intensive/nonintensive chemotherapy in Japanese patients with untreated acute myeloid leukemia or high-risk myelodysplastic syndromes.

Glasdegib is a potent, selective, oral inhibitor of the hedgehog signaling pathway. In this phase I study, previously untreated Japanese patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes were treated with glasdegib (100 mg once daily) combinations: low-dose cytarabine (20 mg twice daily; cohort 1, n = 6; expansion cohort, n = 15); daunorubicin and cytarabine (60 mg/m2 i.v.; cohort 2, n = 6); or azacitidine (100 mg/m2 i.v.; cohort 3, n = 6). Patients, except cohort 2, were ineligible for intensive chemotherapy. The primary end-point was dose-limiting toxicity in cohorts 1-3 and disease-modifying response in the expansion cohort. Disease-modifying response rate was tested with the null hypothesis of 6.8%, which was set based on the results from the phase II BRIGHT AML 1003 study (NCT01546038). No dose-limiting toxicities were observed in cohorts 1 or 3; one patient in cohort 2 experienced a dose-limiting toxicity of grade 3 erythroderma. The most common grade ≥3 treatment-related adverse events were neutropenia and thrombocytopenia (66.7% each) in cohort 1 and thrombocytopenia (60.0%) in the expansion cohort. In the expansion cohort, the disease-modifying response rate was 46.7% (90% confidence interval, 24.4-70.0; p < 0.0001), with all patients achieving either a complete response or complete response with incomplete blood count recovery. Median overall survival was 13.9 months. In this study, the primary disease-modifying response end-point with glasdegib plus low-dose cytarabine was met. The study confirms the safety and efficacy of glasdegib plus low-dose cytarabine in Japanese patients with AML ineligible for intensive chemotherapy.