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PURPOSE To evaluate the safety and efficacy of N-acetylmannosamine (ManNAc) in GNE myopathy, a genetic muscle disease caused by deficiency of the rate-limiting enzyme in N-acetylneuraminic acid (Neu5Ac) biosynthesis. METHODS We conducted an open-label, phase 2, single-center (NIH, USA) study to evaluate oral ManNAc in 12 patients with GNE myopathy (ClinicalTrials.gov NCT02346461). Primary endpoints were safety and biochemical efficacy as determined by change in plasma Neu5Ac and sarcolemmal sialylation. Clinical efficacy was evaluated using secondary outcome measures as part of study extensions, and a disease progression model (GNE-DPM) was tested as an efficacy analysis method. RESULTS Most drug-related adverse events were gastrointestinal, and there were no serious adverse events. Increased plasma Neu5Ac (+2,159 nmol/L, p

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder typically characterized by insoluble inclusions of hyperphosphorylated TDP-43. The mechanisms underlying toxic TDP-43 accumulation are not understood. Persistent activation of p38 mitogen-activated protein kinase (MAPK) is implicated in ALS. However, it is unclear how p38 MAPK affects TDP-43 proteinopathy. Here, we demonstrate that inhibition of p38α MAPK reduces pathological TDP-43 phosphorylation, aggregation, cytoplasmic mislocalization, and neurotoxicity. We establish that p38α MAPK phosphorylates TDP-43 at pathological serine 409/410 (S409/S410) and serine 292 (S292), which reduces TDP-43 liquid-liquid phase separation (LLPS) but allows pathological TDP-43 aggregation. Moreover, we show that protein arginine methyltransferase 1 methylates TDP-43 at R293. Importantly, S292 phosphorylation reduces R293 methylation, and R293 methylation reduces S409/S410 phosphorylation. R293 methylation permits TDP-43 LLPS and reduces pathological TDP-43 aggregation. Thus, strategies to reduce p38α-mediated TDP-43 phosphorylation and promote R293 methylation could have therapeutic utility for ALS and related TDP-43 proteinopathies.

Introduction: CFT7455 is a highly potent and novel Ikaros Family Zinc Finger Protein 1/3 (IKZF1/3) degrader. In xenograft models, CFT7455 has more potent IKZF1/3 degradation compared to other degraders. Early observations from the FIH clinical trial (NCT04756726) along with supporting translational studies are presented here. Methods: Pre-clinical studies comparing CFT7455 and CC-92480 in both in vitro and xenograft models were performed. The pre-clinical studies’ results coincided with observations from the on-going clinical trial. The clinical trial is an open-label, Phase 1/2, multi-center, FIH study in heavily pretreated relapsed/refractory (R/R) MM and non-Hodgkin’s lymphoma (NHL) patients evaluating safety, tolerability, and PK of CFT7455. Eligible MM patients are R/R to therapy and are not candidates for regimens known to provide clinical benefit. A starting dose of 50 μg QD 21 days on/7 days off (21/7) in 28-day cycles was administered. Results: CFT7455 and CC-92480 showed similar cereblon binding profiles and in vitro IKZF1/3 degradation kinetics, translating into sub-nanomolar GI50 values in proliferation assays across a panel of MM cell lines. In the NCI-H929 xenograft model, 100 μg/kg/day of CFT7455 resulted in durable tumor regressions, while 1000 μg/kg/day of CC-92480 gave tumor stasis. Similar results were seen in a systemic model of MM, MM1.S. Both compounds achieved >95% IKZF3 degradation in tumors 4h post dose. At 48h post dose, CFT7455 was more effective than CC-92480 in maintaining IKZF3 degradation (65% vs. 6% respectively). When levels of CFT7455 and CC-92480 in plasma and tumor were compared, CFT7455 concentrations were > DC80 in tumor 48h post dose, while CC-92480 levels were undetectable in tumor and plasma, demonstrating CFT7455 has longer exposure resulting in sustained IKZF1/3 degradation in pre-clinical models. In cohort A, 5 heavily pre-treated MM patients (pts) received single agent CFT7455. 4 pts have received at least 3 cycles, with 2 pts receiving 5 cycles. Neutropenia (grade 4) was observed in 3/5 pts without coincident fever or infection. Additionally, a 2-4 fold accumulation in plasma CFT7455 exposure at steady state was observed. Early pharmacodynamic (PD) data demonstrates deep persistent degradation of IKZF3 (~100%) and serum free light chain reduction (up to 72%) in response to treatment. Stable disease has been observed in 34 pts, suggesting clinical benefit. Conclusions: While CFT7455 showed clinical benefit at 50 ug with deep target degradation, neutropenia was dose limiting. PK/PD modeling suggests alternative dosing regimens may result in increased tolerability with preserved efficacy, and evaluation of them is underway. Updated results will be presented at the meeting. Citation Format: Sagar Lonial, Shambavi Richard, Jeffrey V Matous, Andrew J. Yee, Urvi Shah, Neha Mehta-Shah, Thomas Martin, Eli Muchtar, Sikander Ailawadhi, Paul G. Richardson, Manisha Bhutani, Samantha Perino, Jason Kirby, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, Matthew Schnaderbeck, Michael R. Palmer, Cathleen Gorman, Oliver Schoenborn-Kellenberger, Amanda Hoerres, Stewart L. Fisher, Roy M. Pollock, Adam Crystal, Michelle Mahler, Jesus Bardeja. Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT186.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Background GNE myopathy is a rare genetic disease characterized by progressive muscle atrophy and weakness. It is caused by biallelic mutations in the GNE gene that encodes for the bifunctional enzyme, uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase. Typical characteristics of GNE myopathy include progressive myopathy, first involving anterior tibialis muscle and sparing the quadriceps, and rimmed vacuoles on muscle biopsy. Identifying biallelic mutations by sequencing of the GNE gene confirms the diagnosis of GNE myopathy. In a subset of patients, diagnostic confirmation is challenged by the identification of mutations in only one allele, suggesting mutations in deep intronic regions or regulatory regions. Methods We performed targeted sequencing and copy number variant (CNV) analysis of GNE in two siblings who clinically presented with GNE myopathy. Further molecular and biochemical studies were done to characterize the effect of a previously uncharacterized GNE mutation. Results We report two siblings of Indian descent with characteristic features of GNE myopathy, including progressive skeletal muscle weakness initially involving the anterior tibialis, and rimmed vacuoles on muscle biopsy, in which a heterozygous mutation, p.Val727Met, was identified in both affected siblings, but no other deleterious variants in either coding region or exon–intron boundaries of the gene. Subsequent insertion/deletion analysis identified a novel 11.3-kb deletion (Chr9 [GRCh37]: g.36257583_36268910del) encompassing the GNE promoter region, with breakpoints residing in Alu repeats. Gene expression analysis revealed reduced GNE mRNA and protein levels, confirming decreased expression of the deleted allele harboring the deletion. Conclusions We have identified GNE as one of the genes susceptible to Alu-mediated recombination. Our findings suggest that the deletion may encompass the promoter or another region necessary for GNE expression. In patients with typical manifestations of GNE myopathy and a single GNE variant identified, copy number variant (CNV) analysis may be useful in arriving at the diagnosis.

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.

Introduction Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 in MM cells inhibits growth, confirming their dependency on IKZF1/3. IMiDs (lenalidomide[len], pomalidomide[pom]) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. Most patients treated with len or pom eventually develop progressive disease due to acquired resistance, underscoring an unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high affinity cereblon (CRBN) binding, rapid IKZF1/3 degradation, and potent in vivo efficacy. Results CFT7455 demonstrated an 800 to 1600-fold improvement in CRBN binding compared to pom in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hr. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines as well as H929 cells made resistant to IMiDs. In RPMI-8226 MM mice xenografts, CFT7455 (0.1 mg/kg/day) resulted in deep, durable degradation of IKZF3 (21% and 9.5% of vehicle levels at 4 and 24 hr, respectively). Protein levels for IRF4, an essential TF in MM, declined over 7 days with daily CFT7455 treatment to 8% of vehicle levels. Dose dependent efficacy ranged from 0.003-0.1 mg/kg/day, with tumor regression evident at doses ≥0.01 mg/kg/day. Pom was inactive in this model at a human equivalent dose (3 mg/kg/day), with no observed tumor shrinkage in these mice following 17 days of dosing. Switching pom treatment to CFT7455 (0.1 mg/kg/day) on Day 18 led to tumor regression in 67% of animals on Day 28 and 100% tumor regression on Day 35, demonstrating that CFT7455 penetrates large tissues and is efficacious in rapidly growing, IMiD resistant tumors. In the H929 tumor xenograft model, administration of CFT7455 (0.1 mg/kg/day) promoted tumor regression (95% tumor growth inhibition by 7 days); dosing was stopped after 21 days. On Day 63, half the tumors remained below their starting tumor volume. Additionally, CFT7455 demonstrated durable tumor regression in the aggressive MM1.S systemic MM tumor model. In mice bearing RPMI-8226 xenograft tumors, the combination of CFT7455 (QD) and dexamethasone (QW) was more active, and demonstrated a significant survival improvement, compared to either agent alone. Conclusions CFT7455 is a highly potent, catalytic degrader of IKZF1/3, with marked antitumor activity as a single agent and in combination with dexamethasone. Importantly, CFT7455 retains activity in models resistant or insensitive to IMiDs. These results warrant investigation of CFT7455 as a therapeutic approach for MM and a clinical study is planned. Citation Format: James A. Henderson, R. Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Scott J. Eron, Andrew Good, Ashley A. Hart, Christina Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Roy M. Pollock, Adam S. Crystal, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB007.

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress., Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. Significance Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.

Introduction GNE myopathy is an adult-onset muscle disorder characterized by impaired sialylation of (muscle) glycans, detectable by lectin histochemistry. We describe a standardized method to quantify (lectin-) fluorescence in muscle sections, applicable for diagnosis and response to therapy for GNE myopathy. Methods Muscle sections were fluorescently labeled with the sialic acid-binding Sambucus nigra agglutinin (SNA) lectin and antibodies to sarcolemma residence protein caveolin-3 (CAV-3). Entire tissue sections were imaged in tiles and fluorescence was quantified. Results SNA fluorescence co-localizing with CAV-3 was ∼50% decreased in GNE myopathy biopsies compared with muscle-matched controls, confirming previous qualitative results. Discussion This quantitative fluorescence method can accurately determine sialylation status of GNE myopathy muscle biopsies. This method is adaptable for expression of other membrane-associated muscle proteins, and may be of benefit for disorders in which therapeutic changes in expression are subtle and difficult to assess by other methods. Muscle Nerve 58: 286-292, 2018.

Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

Background GNE myopathy is a rare genetic disease characterized by progressive muscle atrophy and weakness. It is caused by biallelic mutations in the GNE gene that encodes for the bifunctional enzyme, uridine diphosphate (UDP)‐N‐acetylglucosamine (GlcNAc) 2‐epimerase/N‐acetylmannosamine (ManNAc) kinase. Typical characteristics of GNE myopathy include progressive myopathy, first involving anterior tibialis muscle and sparing the quadriceps, and rimmed vacuoles on muscle biopsy. Identifying biallelic mutations by sequencing of the GNE gene confirms the diagnosis of GNE myopathy. In a subset of patients, diagnostic confirmation is challenged by the identification of mutations in only one allele, suggesting mutations in deep intronic regions or regulatory regions. Methods We performed targeted sequencing and copy number variant (CNV) analysis of GNE in two siblings who clinically presented with GNE myopathy. Further molecular and biochemical studies were done to characterize the effect of a previously uncharacterized GNE mutation. Results We report two siblings of Indian descent with characteristic features of GNE myopathy, including progressive skeletal muscle weakness initially involving the anterior tibialis, and rimmed vacuoles on muscle biopsy, in which a heterozygous mutation, p.Val727Met, was identified in both affected siblings, but no other deleterious variants in either coding region or exon–intron boundaries of the gene. Subsequent insertion/deletion analysis identified a novel 11.3‐kb deletion (Chr9 [GRCh37]: g.36257583_36268910del) encompassing the GNE promoter region, with breakpoints residing in Alu repeats. Gene expression analysis revealed reduced GNE mRNA and protein levels, confirming decreased expression of the deleted allele harboring the deletion. Conclusions We have identified GNE as one of the genes susceptible to Alu‐mediated recombination. Our findings suggest that the deletion may encompass the promoter or another region necessary for GNE expression. In patients with typical manifestations of GNE myopathy and a single GNE variant identified, copy number variant (CNV) analysis may be useful in arriving at the diagnosis.