Apilimod dimesylate in C9orf72 amyotrophic lateral sclerosis: a randomized phase 2a clinical trial.

Apilimod dimesylate is a first-in-class phosphoinositide kinase, FYVE-type zinc finger-containing (PIKfyve) inhibitor with a favourable clinical safety profile and has demonstrated activity in preclinical C9orf72 and TDP-43 amyotrophic lateral sclerosis (ALS) models. In this ALS clinical trial, the safety, tolerability, CNS penetrance and modulation of pharmacodynamic target engagement biomarkers were evaluated. This phase 2a, randomized, double-blind, placebo-controlled, biomarker-end-point clinical trial was conducted in four US centres (ClinicalTrials.gov NCT05163886). Participants with C9orf72 repeat expansions were randomly assigned (2:1) to receive twice-daily oral treatment with 125 mg apilimod dimesylate capsules or matching placebo for 12 weeks, followed by a 12-week open-label extension. Safety was measured as the occurrence of treatment-emergent or serious adverse events attributable to the study drug and tolerability at trial completion or treatment over 12 weeks. Changes from baseline in plasma and CSF and concentrations of apilimod dimesylate and its active metabolites and of pharmacodynamic biomarkers of PIKfyve inhibition [soluble glycoprotein nonmetastatic melanoma protein B (sGPNMB) upregulation] and disease-specific CNS target engagement [poly(GP)] were measured. Between 16 December 2021 and 7 July 2022, 15 eligible participants were enrolled. There were no drug-related serious adverse events reported in the trial. Fourteen (93%) participants completed the double-blind period with 99% dose compliance [n = 9 (90%) apilimod dimesylate; n = 5 (100%) placebo]. At Week 12, apilimod dimesylate was measurable in CSF at 1.63 ng/ml [standard deviation (SD): 0.937]. At Week 12, apilimod dimesylate increased plasma sGPNMB by >2.5-fold (P < 0.001), indicating PIKfyve inhibition, and lowered CSF poly(GP) protein levels by 73% (P < 0.001), indicating CNS tissue-level proof of mechanism. Apilimod dimesylate met prespecified key safety and biomarker end-points in this phase 2a trial and demonstrated CNS penetrance and pharmacodynamic target engagement. Apilimod dimesylate was observed to result in the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.

LAM-003, a new drug for treatment of tyrosine kinase inhibitor-resistant FLT3-ITD-positive AML.

Acute myeloid leukemias (AML) harboring a constitutively active internal tandem duplication (ITD) mutation in the FMS-like kinase tyrosine kinase (FLT3) receptor are associated with poor patient prognosis. Despite initial clinical responses to FLT3 kinase inhibitors, patients eventually relapse. Mechanisms of resistance include the acquisition of secondary FLT3 mutations and protective stromal signaling within the bone marrow niche. Here we show that LAM-003, a prodrug of the heat shock protein 90 inhibitor LAM-003A, has cytotoxic activity against AML cell lines and primary samples harboring FLT3-ITD. LAM-003 regressed tumors in an MV-4-11 xenograft mouse model and extended survival in a MOLM-13 systemic model. LAM-003 displayed synergistic activity with chemotherapeutic drugs and FLT3 inhibitors, with the most robust synergy being obtained with venetoclax, a BCL-2 inhibitor. This finding was verified in a MOLM-13 systemic survival model in which the combination significantly prolonged survival compared with the single agents. Importantly, LAM-003 exhibited equipotent activity against FLT3 inhibitor-resistant mutants of FLT3, such as D835 or F691, in cytotoxic and FLT3 degradation assays. LAM-003 also retained potency in AML cells grown in stromal-conditioned media that were resistant to FLT3 inhibitors. Lastly, a genome-wide CRISPR screen revealed epigenetic regulators, including KDM6A, as determinants of LAM-003 sensitivity in AML cell lines, leading to the discovery of synergy with an EZH2 inhibitor. Collectively, these preclinical findings support the use of LAM-003 in FLT3-ITD patients with AML who no longer respond to FLT3 inhibitor therapy either as a single agent or in combination with drugs known to be active in AML.

Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine.

BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

Discovery of a new chemical series of BRD4(1) inhibitors using protein-ligand docking and structure-guided design.

Bromodomains are key transcriptional regulators that are thought to be druggable epigenetic targets for cancer, inflammation, diabetes and cardiovascular therapeutics. Of particular importance is the first of two bromodomains in bromodomain containing 4 protein (BRD4(1)). Protein-ligand docking in BRD4(1) was used to purchase a small, focused screening set of compounds possessing a large variety of core structures. Within this set, a small number of weak hits each contained a dihydroquinoxalinone ring system. We purchased other analogs with this ring system and further validated the new hit series and obtained improvement in binding inhibition. Limited exploration by new analog synthesis showed that the binding inhibition in a FRET assay could be improved to the low µM level making this new core a potential hit-to-lead series. Additionally, the predicted geometries of the initial hit and an improved analog were confirmed by X-ray co-crystallography with BRD4(1).

Peginesatide and erythropoietin stimulate similar erythropoietin receptor-mediated signal transduction and gene induction events.

Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. After stimulation with peginesatide or rHuEPO, cell lysates were prepared from UT-7/EPO cells. Liquid chromatography-tandem mass spectrometry and MesoScale arrays were used to quantify phosphorylation events. Transcriptional changes were analyzed using microarrays and quantitative reverse transcription polymerase chain reaction. Peginesatide and rHuEPO were found to regulate the tyrosine phosphorylation of an essentially equivalent set of protein substrates, and modulate the expression of a similar set of target genes. Consistent with their roles in stimulating erythropoiesis, peginesatide and rHuEPO regulate similar cellular pathways.

Dynamic ligand modulation of EPO receptor pools, and dysregulation by polycythemia-associated EPOR alleles.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.

A novel automated assay for the rapid identification of metastatic breast carcinoma in sentinel lymph nodes.

BACKGROUND: The authors prospectively evaluated the performance of a proprietary molecular testing platform using one-step nucleic acid amplification (OSNA) for the detection of metastatic carcinoma in sentinel lymph nodes (SLNs) in a large multicenter trial and compared the OSNA results with the results from a detailed postoperative histopathologic evaluation (reference pathology) and from intraoperative imprint cytology (IC). METHODS: In total, 1044 SLN samples from 496 patients at 11 clinical sites were analyzed. Alternate 1-mm sections were subjected to either detailed histopathologic evaluation with hematoxylin and eosin and pancytokeratin immunostaining or the OSNA Breast Cancer System, which was calibrated to detect tumor deposits >0.2 mm by measuring cytokeratin 19 messenger RNA. At 7 sites, IC was performed before permanent section. The OSNA results were classified as negative (<250 copies/µL), micrometastases (from ≥250 to <5000 copies/µL), or macrometastases (≥5000 copies/µL). RESULTS: The sensitivity and specificity of the OSNA breast cancer system compared with reference pathology were 77.5% (95% confidence interval, 69.7%-84.2%) and 95.8% (95% confidence interval, 94.3%-97.0%), respectively, before discordant case analyses (DCA). Sensitivity and specificity after DCA were 82.7% and 97.7%, and final concordance was 95.8%. Performance for invasive lobular carcinoma demonstrated 88.2% sensitivity (95% confidence interval, 63.6%-98.5%) and 98.5% specificity (95% confidence interval, 92%-100%). The sensitivity of OSNA was significantly better than that of IC (80% vs 63%; P = .0229). CONCLUSIONS: The OSNA breast cancer system proved to be highly accurate for the detection of metastatic breast cancer in axillary SLNs. Sensitivity was comparable to that predicted for conventional postoperative histologic examination at 2-mm intervals and was significantly more sensitive than IC. Automation, semiquantitative results enabling the differentiation of macrometastasis and micrometastasis, and rapid results render the assay suitable for intraoperative and/or permanent evaluation of SLNs.

Evaluation of antitumor properties of novel saframycin analogs in vitro and in vivo.

Novel analogs of (-)-saframycin A are described. The analogs are shown to be potent inhibitors of the in vitro growth of several tumor cells in a broad panel and promising as leads for further optimization. The first in vivo studies in a solid tumor model (HCT-116) reveal potent antitumor activity with associated toxicity of daily administration.

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo.

CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K(i) of 0.007 mumol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.

Intraoperative imprint cytologic evaluation of sentinel lymph nodes for lobular carcinoma of the breast.

BACKGROUND: The evaluation of sentinel lymph nodes (SLNs) from a woman with lobular cancer of the breast is frequently challenging. Intraoperative imprint cytology (IIC) is equivalent to frozen sectioning for rapid SLN evaluation and is advantageous because it is rapid, reliable, cost-effective, and conserves tissue. Metastatic lobular carcinoma is difficult to identify in SLN because of its low-grade cytomorphology, its tendency to infiltrate lymph nodes in a single cell pattern, and because individual cells can resemble lymphocytes. We are unaware of any large published studies, using any technique, to evaluate SLN for lobular carcinoma. METHODS: A retrospective review of the intraoperative imprint cytology results of 678 SLN mapping procedures for breast carcinoma was performed. From this cohort, we studied SLN from cases of lobular carcinoma. These SLN were evaluated intraoperatively by either bisecting or slicing the SLN into 4-mm sections. Imprints were made of each cut surface and stained with hematoxylin and eosin and/or Diff-Quik. Permanent sections were evaluated with up to 4 hematoxylin and eosin-stained levels and cytokeratin immunohistochemistry. IIC results were compared with final histologic results. RESULTS: Sixty-one cases of pure invasive lobular carcinoma were identified. Sensitivity was 52%, specificity was 100%, accuracy was 82%, negative predictive value was 78%. No statistically significant differences in sensitivity, specificity or accuracy were identified for the intraoperative detection of lobular carcinoma versus ductal carcinoma. The sensitivity for detecting macrometastases (more than 2 mm) was better than for detecting micrometastases, 73 versus 25%, respectively (P = 0.059). CONCLUSIONS: The sensitivity and specificity of IIC are similar to that of intraoperative frozen section evaluation. Therefore, IIC is a viable alternative to frozen sectioning when intraoperative evaluation is required. If SLN micrometastasis is used to determine the need for further lymphadenectomy, more sensitive intraoperative methods will be needed to avoid a second operation.

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Intraoperative evaluation of sentinel lymph nodes for metastatic breast carcinoma by imprint cytology.

BACKGROUND: The increasing utilization of lymphatic mapping techniques for breast carcinoma has made intraoperative evaluation of sentinel lymph nodes attractive. Axillary lymph node dissection can be performed during the initial surgery if the sentinel lymph node is positive, potentially avoiding a second operative procedure. At present the optimal technique for rapid sentinel lymph node assessment has not been determined. Both frozen sectioning and intraoperative imprint cytology are used for rapid intraoperative sentinel lymph node evaluation at many institutions. The purpose of this study is to evaluate experience with imprint cytology for intraoperative evaluation of sentinel lymph nodes in patients with breast cancer. METHODS: A retrospective review of the intraoperative imprint cytology results of 678 sentinel lymph node mappings for breast carcinoma was performed. Sentinel nodes were evaluated intraoperatively by either bisecting or slicing the sentinel node into 4 mm sections. Imprints were made of each cut surface and stained with H&E and/or Diff-Quik. Permanent sections were evaluated with up to four H&E stained levels and cytokeratin immunohistochemistry. Intraoperative imprint cytology results were compared with final histologic results. RESULTS: The sensitivity of imprint cytology was 53%, specificity was 98%, positive predictive value was 94%, negative predictive value was 82% and accuracy was 84%. The sensitivity for detecting macrometastases (more than 2mm) was significantly better than for detecting micrometastases (

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice.

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2(-/-)) mice. MK2(-/-) mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2(-/-) mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1beta mRNA (53% reduction) but not in tumor necrosis factor-alpha mRNA in MK2(-/-) mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1beta was also confirmed in MK2(-/-) mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2(-/-) mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1beta in the ischemic brain tissue.

Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor alpha.

The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331

Generation of multiple farnesoid-X-receptor isoforms through the use of alternative promoters.

Bile acid biosynthesis is regulated by both feed-forward and feedback mechanisms involving a cascade of nuclear hormone receptors. Feed-forward regulation of the rate limiting enzyme in bile acid biosynthesis is provided by oxysterols through liver-X-receptor alpha (NR1H3), while feedback regulation is provided by bile acids through farnesoid-X-receptor (FXR) (NR1H4). The Syrian golden hamster provides a useful model for studying lipid metabolism. The hamster metabolizes and transports dietary cholesterol in a similar manner to humans, with the resulting lipid profile being more similar to the human profile than that of other rodent models. Cloning of Fxr from Syrian golden hamster revealed four hamster Fxr splice variants that altered the N-terminal activation domain or the hinge region between the DNA and ligand binding domains. Human genomic sequence and data from hamster Fxr were used to identify and clone a novel human FXR isoform resulting from the use of an alternative promoter. RNA expression analysis indicates that the two human FXR isoforms are differentially expressed in developmental and tissue-specific patterns and are likely to provide a mechanism for cell-specific FXR-dependent transcriptional activity.

Intraoperative evaluation of lumpectomy margins by imprint cytology with histologic correlation: a community hospital experience.

BACKGROUND: Several well-controlled studies have demonstrated significantly increased local recurrence rates in patients with low-stage breast carcinoma treated with breast conservation therapy in whom focally positive margins were not reexcised. Imprint cytology is a rapid technique for evaluating surgical margins intraoperatively, thus allowing reexcisions to be performed during the initial surgery. The large majority of studies on the use of intraoperative imprint cytologic examination of breast conservation therapy margins have been performed at university-based academic centers. OBJECTIVE: To evaluate the utility of intraoperative imprint cytologic evaluation of breast conservation therapy margins in a community hospital setting. METHODS: We retrospectively reviewed the intraoperative imprint cytology margins of 141 lumpectomy specimens that had been obtained from 137 patients between May 1997 and May 2001. RESULTS: We evaluated 758 separate margins. On a patient basis, the sensitivity was 80%, the specificity was 85%, the positive predictive value was 40%, the negative predictive value was 97%, and the overall accuracy was 85%. There were no cytologically unsatisfactory margins. CONCLUSION: Imprint cytology is an accurate, simple, rapid, and cost-effective method for determining the margin status of breast conservation therapy specimens intraoperatively in the community hospital setting. This method allows a survey of the entire surface area of the lumpectomy specimen, which is not practical using frozen section evaluation.