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Abstract: Mutations in calreticulin (mutCALR) are the second most common drivers of myeloproliferative neoplasms (MPNs) and yet, the current therapeutic landscape lacks a selective agent for mutCALR-expressing MPNs. Here, we show that the monoclonal antibody INCA033989 selectively targets mutCALR-positive cells. INCA033989 antagonized mutCALR-driven signaling and proliferation in engineered cell lines and primary CD34+ cells from patients with MPN. No antibody binding or functional activity was observed in the cells lacking mutCALR. In a mouse model of mutCALR-driven MPN, treatment with an INCA033989 mouse surrogate antibody effectively prevented the development of thrombocytosis and accumulation of megakaryocytes in the bone marrow. INCA033989 reduced the pathogenic self-renewal of mutCALR-positive disease-initiating cells in both primary and secondary transplantations, illustrating its disease-modifying potential. In summary, we describe a novel mutCALR-targeted therapy for MPNs, a monoclonal antibody that selectively inhibits the oncogenic function of MPN cells without interfering with normal hematopoiesis., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies., Significance: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397., (©2022 The Authors; Published by the American Association for Cancer Research.)

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations., Competing Interests: Holly Koblish, Liangxing Wu, Kevin Bowman, Sharon Diamond, Darlise DiMatteo, Yue Zhang, Michael Hansbury, Mark Rupar, Xiaoming Wen, Paul Collier, Patricia Feldman, Ronald Klabe, Krista A. Burke, Maxim Soloviev, Christine Gardiner, Xin He, Alla Volgina, Maryanne Covington, Richard Wynn, Timothy C. Burn, Swamy Yeleswaram, Wenqing Yao are employees of Incyte Corporationand own Incyte stock. Phillip C. C. Liu, Bruce Ruggeri, Peggy Scherle, Reid Huber, and Gregory Hollis were employees of Incyte employees Corporation at the time of this work and own Incyte stock.Our commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose: Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials., Experimental Design: We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations., Results: In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo . In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK-STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo . This combination potentiated tumor growth inhibition in vivo , even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone., Conclusions: Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329., (©2018 American Association for Cancer Research.)

Background: Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation., Results: Single-stranded DNA oligonucleotides that were designed to repair this splice site mutation corrected the mutation in the gene and restored expression of wild-type dystrophin. This repair was validated at the DNA, RNA and protein level. We also report that the frequency of genetic repair of the mdx mutation can be enhanced if RNAi is used to suppress expression of the recombinase inhibitor protein Msh2 in cultures containing myoblasts but not in those heavily enriched in myoblasts., Conclusion: Exogenous manipulations, such as RNAi, are certainly feasible and possibly required to increase the successful application of gene repair in some primary or progenitor muscle cells.

Spinal muscular atrophy (SMA), a recessive, neuromuscular disease, is caused by a mutation or deletion in the SMN1 gene. The SMN2 gene is present in the same region of chromosome 5 and is similar in DNA sequence to SMN1 except for a T at position +6 of exon 7 that is likely the predominant functional difference between the two genes. This change alters RNA splicing which results in the removal of exon 7 from the mature mRNA; only 10% full-length transcripts are produced from the SMN2 gene. Our lab has shown that single-stranded oligonucleotides (ODN) can be used to repair genes with single base mutations within the context of the native chromosome. Here, we used SMN2-sequence-specific ODNs to direct the exchange of a T to a C in an SMA skin fibroblast cell line from a type 1 patient. The cells were transfected with ODNs of either 47 or 75 bases in length and designed to hybridize to either the transcribed or non-transcribed DNA strand of the SMN2 gene. We analyzed the genotype of these cells using a well-established Taqman probe-based PCR assay, restriction enzyme digestion, and cycle sequencing. Conversion of the SMN2 genotype to SMN1 was detected when the specific ODN was added. As a result, we observed an increase in production of full-length SMN mRNA, measured by qRT-PCR, and SMN protein, measured by western blotting. Finally, properly localized SMN protein was detected by the accretion of gemini of coiled bodies (gems) only in targeted cells. This is the first report of the use of ODNs to direct genetic conversion of SMN2 to SMN1 in human cells from SMA patients.

Skeletal metastases are often complicated by progression to impending or pathologic fracture and fixation with polymethylmethacrylate (PMMA) bone cement is used for stabilization and pain relief. Adjuvant therapy involving the delivery of PMMA composites mixed with antibiotic or chemotherapeutic agents requires an understanding of the rate of drug diffusion from the cement in addition to measurement of its mechanical properties pre- and postelution of drug. We have developed a method for the analysis of drug diffusion rate and mechanical properties of drug-cement composites using PMMA/methotrexate as a model system. The analysis method revealed the addition of methotrexate to PMMA in concentrations of 1.8 g methotrexate per 40 g PMMA did not change the compression modulus of the cement pre- or postelution of drug. The PMMA/methotrexate composites displayed an average diffusion rate of 50 ng/(mm2)(hour) during the first 6 hours, which decreased to 10 ng/(mm2)(hour) by 36 hours. Diffusion modeling predicts the 20 x 13-mm cylindrical PMMA/methotrexate samples used by the method deliver 10% of the total methotrexate content within 80 hours and 25% of the total within 133 days.

Pulmonary alveolar type II cells synthesize and secrete phospholipids and surfactant proteins. In most mammalian species, the synthesis of phospholipids and proteins of lung surfactant increases with fetal lung maturation, which occurs late in gestation. Factors that may promote lung maturation and surfactant production include the placental hormone, leptin, whose expression increases with advancing gestational age. We demonstrate that physiologic concentrations of leptin (1 and 10 ng/mL) increase the levels of surfactant proteins (SP) A, B, and C mRNA as well as SP-A and SP-B protein in d-17 fetal rat lung explants in vitro. To determine whether leptin exerts similar effects in vivo, we administered leptin antenatally to pregnant rats and compared its effects to that of dexamethasone, a known mediator of fetal lung development. Antenatal treatment with leptin for 2 d significantly increased the average weight of the fetal lungs in relation to their body weight. Histologic analysis revealed that the increase in fetal lung weight was accompanied by an increase in the number and maturation of type II alveolar cells and the expression of surfactant proteins B and C in these cells. Collectively, these results suggest that leptin is a cytokine regulator of rat fetal lung maturity.

Purpose: To determine whether maternal age, race, or income, is the primary determinant of breastfeeding continuation rates., Methods: Using the Delaware modified-PRAMS 2000 de-identified data set, multivariate modeling of age, income, race, weight, health status, and other breastfeeding continuation factors were analyzed., Results: Breastfeeding continuation at 12 weeks of age was predicted by maternal age independent of race or income. Mothers greater than the mean age were 86% more likely to continue to breastfeed than younger mothers (OR 1.861 95% CI 1.234-2.805). Maternal BMI > or = 26, nipple soreness, and breastfeeding difficulty were associated with lower breastfeeding continuation., Discussion: Maternal age, not race or income, are driving factors in breastfeeding continuation. Physiologic variables, such as maternal obesity, breastfeeding difficulty, and nipple soreness have an adverse influence on duration of breastfeeding. Investment into processes that lengthen breastfeeding duration among mothers, especially younger mothers, who chose to initiate breastfeeding, may have individual and community benefits.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the telomeric copy of the survival motor neuron (SMN1) gene. Although the SMN protein has been implicated in the biogenesis of ribonucleoprotein complexes and RNA processing, it is not clear how these functions contribute to the pathogenesis of SMA. To gain a further understanding of SMN function, we have investigated its role in cell survival in skin fibroblasts derived from SMA patients and age-matched controls. SMA fibroblasts exposed to camptothecin, a specific inhibitor of DNA topoisomerase I, consistently showed cell death at a lower concentration than normal controls. Treatment with other cell death-inducing agents did not cause differences in survival of SMA fibroblasts as compared with control fibroblasts. Camptothecin treatment resulted in activation of caspase-3 with generation of the caspase-3 cleavage product, poly ADP-ribose polymerase (PARP). Depletion of SMN protein by RNA interference in control fibroblasts increased caspase-3 activity, whereas transfection of SMA fibroblasts with wild-type SMN decreased caspase-3 activity. Our data demonstrate that SMA fibroblasts are more prone to some, but not all, death-stimuli. Vulnerability to death-stimuli is associated with decreased levels of SMN protein and is mediated by activation of caspase-3.