Reduction In Splenomegaly Research Articles

Durable Clinical Benefits of Idasanutlin Therapy in Hydroxyurea-Refractory Polycythemia Vera

Background: In phase 1 (NCT02407080) and phase 2 (NCT03287245) clinical trials, idasanutlin, a human double minute 2 homolog (HDM2) antagonist, demonstrated robust clinical activity and rapid reduction in JAK2V617F variant allele frequencies (VAF) in patients with hydroxyurea-intolerant or refractory polycythemia vera (PV). Despite promising disease-targeted clinical efficacy, high rates of discontinuation due to toxicity, and concerns regarding the potential clinical implications of transient TP53 mutations had dampened the enthusiasm for pursuing further development of this class of drugs in PV patients. 1,2,3 Given the significant reduction in JAK2V617F VAF that was documented in the clinical trials, we postulated that idasanutlin may have durable clinical effects in terms of reduction in phlebotomy requirement, splenomegaly, and disease progression, as well as questioned the significance of the transient increments in TP53 mutations. To assess the long-term implications of this therapy on PV patients, we examined the longer-term effects of idasanutlin administration. Methods: We retrospectively evaluated the long-term outcomes of patients enrolled in the phase 1 and 2 clinical trials and used descriptive statistical analysis to evaluate a change in phlebotomy requirements, change in spleen size, and JAK2V617F VAF. Results: Seventy-six percent (29/38) of the patients enrolled in the phase 1 and phase 2 studies continued to be followed at the participating institutions after the end of trial (EOT) visit (Table 1). Median follow-up after EOT to last follow up visit was 40 months (range XX-XX months). There was no increase in 8-week phlebotomy requirements when comparing 8-week phlebotomy requirement prior to EOT to pre-specified time-points of 1, 3, 6, 12, 18, 24, 36 months after idasanutlin discontinuation (P=.50). Six of 29 (21%) participants remained off PV-directed therapy for the duration of follow up. Therapies initiated in the remainder of the patients are listed in Table 1, four of whom received either combination or sequential therapies. Reduction in splenomegaly with idasanutlin was durable with no observed increase in spleen size by palpation from EOT through the 36-month follow up visit (P=.51). Change in hematologic profile of patients throughout follow up is shown in Figure 1 (median, range). There were four (13.7%) documented cases of disease progression. Three progressed to myelofibrosis (MF), one to blast phase (BP). Two participants that progressed to MF went on to receive allogeneic hematopoietic cell transplant. Of those who progressed, median time to progression was 17.5 months (range 14-24 months) after treatment discontinuation. Of those that progressed, mutations by next generation sequencing were noted in JAK2 (4), TET2 (2), DNMT3A (1), SF3B1 (1), and ASXL1 (1). None had a known TP53 mutation at the time of disease progression. There were no documented cases of thrombotic events, or hemorrhagic events within the follow-up period. Changes in JAK2V617F VAF after treatment discontinuation will be presented at the meeting. Conclusion: HDM2 antagonist therapy represents a rational and highly effective treatment approach in PV. The durable effects of idasanutlin on phlebotomy requirements and splenomegaly persist even after treatment discontinuation. Additionally, the lack of appearance of TP53 mutations or loss of TP53 in those who progress suggest that the previously-described transient TP53 mutations do not have long-term clinical implications. Alternative dose and scheduling approaches that are more tolerable may be warranted to safely maintain long-term PV disease control. 1 Mascarenhas J, Passamonti F, Burbury K, et al. The MDM2 antagonist idasanutlin in patients with polycythemia vera: results from a single-arm phase 2 study. Blood Advances. 2022;6(4):1162-1174. doi:10.1182/bloodadvances.2021006043 2 Mascarenhas J, Lu M, Kosiorek H, et al. Oral idasanutlin in patients with polycythemia vera. Blood. 08 08 2019;134(6):525-533. doi:10.1182/blood.2018893545 3 Marcellino BK, Farnoud N, Cassinat B, et al. Transient expansion of TP53 mutated clones in polycythemia vera patients treated with idasanutlin. Blood Advances. 2020;4(22):5735-5744. doi:10.1182/bloodadvances.2020002379

Single-Cell RNA Sequencing Uncovers Abnormal Differentiation of Neutrophil and Megakaryocyte Might Mediated Ruxolitinib Resistance for Patinets with PMF

Background: Primary myelofibrosis (PMF ) is a heterogeneous disorder characterized by bone marrow fibrosis, megakaryocyte hyperplasia, systemic inflammation and extramedullary hematopoiesis. Ruxolitinib (Rux）have shown clinical benefit with reduction of splenomegaly and related symptoms. However, about 50% of patients discontinued ruxolitinib for resistance and/or intolerance，and the mechanism of resistance to ruxolitinib is still unclear. Recently studies found that immune microenvironment and megakaryocyte promote PMF progression, whether the abnormalities of immune microenvironment and megakaryocyte associated with Rux resistance needs to be further elucidated. M ethods: In this study, 8 participants (Newly Diagnosed PMF =3, Resistance to Rux PMF =3, Healthy Donors =2) were included. Peripheral blood was taken and single-cell transcriptome sequencing technology was used. Combined with pseudo-temporal analysis, SCENIC analysis, Single Cell Lineage Inference Using Cell Expression Similarity and Entropy (SLICE), cellular interaction analysis, gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analysis, Gene Set Variation Analysis (GSVA), L-R receptor coexpression analysis and GEPIA online tool for data analysis, Use R language or Python for data processing. Results: In PMF patients, the proportion of neutrophils and monocytes was significantly increased, while the proportion of T cells was significantly decreased in Resistance to Rux group (P<0.05). Further subdivision of neutrophils showed that neutrophils were divided into 6 subgroups. Compared with healthy control group, neutrophils increased in Resistance to Rux group and decreased in Neutrophil_1, among which Neutrophil_6 was only enriched in PMF patients. Neutrophil_6 significantly up-regulated tumor necrosis factor (TNF) superfamily product pathway, IL-1 signaling pathway, Toll-like Receptor 4 (TLR4) signaling pathway, peroxide-producing NADPH oxidase activator activity pathway, MAPK pathway and IL-8 product signaling pathway. Neutrophil_4-6 significantly up-regulates MYB, EZH2, SUZ12, YBX1, POLR3G, E2F7, E2F8 and other transcription factors. L-R up-regulation between Neutrophil_4 and Neutrophil_6 and T cells mediated T cell exhaustion by LGALS9/Tim3 relate to resistance to Rux. We have identified a subset of HSCs in PMF that exhibits a bias towards megakaryocytic/platelet differentiation (HSCs_2) in Rux resistant group. This subset is characterized by the upregulation of specific genes, such as GATA1, PRKAR2B, ITGA2B, PLEK, and KLF1.CellPhoneDB revealed that the regulation of platelets-platelets involves CD40L-CD40 ligand receptors. Notably, the heightened reactivity of platelets may be closely associated with the risk of PMF-related thrombosis. Additionally, we observed an upregulation of the MIF-CD74 cytokine ligand receptor between the immune platelet subsets and HSC subsets in Rux resistant group. Conclusion: In Rux resistant group, Up-regulation of inflammatory and tumor-related signaling pathways promotes the occurrence of PMF inflammation and amplification of malignant clones; Up-regulation of LGALS9 in neutrophil band to receptor TIM3 mediates T cell depletion; the regulation of platelets-platelets in PMF is mediated by CD40L-CD40 ligand receptors; MIF-CD74 might be the relationship between PMF-HSC and PMF-PLT in the context of primary myelofibrosis.

In Vivo Ablation of NFκB Cascade Effectors Alleviates Disease Burden in Myeloproliferative Neoplasms

ABAL and TK equal contributors Multilevel regulation of NFκB activation exists in response to microenvironmental cues. Aberrant hyperactivation of the NFκB cascade propagates oncogenic signaling and inflammation, which together augment disease burden in myeloproliferative neoplasms (MPNs). Here, we systematically ablated NFκB signaling effectors to identify core dependencies using a series of primary samples and syngeneic and patient-derived xenograft (PDX) mouse models. We first examined the impact of Rela abrogation in the Jak2 V617F knock-in mouse model that exemplifies polycythemia vera hallmarks and generated Jak2V617FRela flox/flox Ubc-Cre-ERT2 ( Jak2- Rela fl) mice. Evident suppression of leukocytosis was observed within a week of Rela excision and sustained until endpoint. Elevated hematocrit, monocytes, and CD45.2 myeloid cells were suppressed in both the peripheral blood (PB) and bone marrow (BM) and Jak2- Rela flmice had significant reduction of splenomegaly. We then adopted a second MPN model, where MPL W515L retroviral expression produces a phenotype resembling myelofibrosis. Consistent reduction of pathologic WBC counts, monocytes, and PB and BM CD45.2 myeloid cells were observed in MPL- Rela fl mice. Rela ablation also reduced hematocrit, platelet counts, and CD45.2 GFP+ cells. Importantly, we observed a significant attenuation of disease hallmarks with a decrease of spleen weight and dysmegakaryopoiesis, enhanced survival, and reduction of BM reticulin deposition. We next established PDX models where CD34+ cells from MF patients and healthy BM donors (NBM) were transduced with an sgRNA targeting RELA and engrafted into NSGS mice. Across two MPN PDXs, there was significant suppression of leukemic cells and improvement of disease burden, reduced spleen weights and prolonged survival, and subsequent BM histology revealing improved sclerosis, fibrosis, and dysmegakaryopoiesis. In contrast, NSGS mice engrafted with NBM CD34+ cells did not yield disease symptoms, nor did RELA targeting in healthy cells affect hematopoiesis or measured disease hallmarks. RNA-seq on CD34+ cells from four MPN patients following RELA abrogation revealed augmented and altered expression of genes that are 1) regulators of survival and proliferative pathways: MDM2, TP53INP1, and FAS 2) major ferroptosis mediator GPX4, and 3) cell cycle effectors: CDKN1A (p21), E2F4, and CHEK2. In further investigation of NFκB pathway effectors, we genetically targeted upstream effector Myd88 and transduced Kit+ cells from Myd88-null mice with MPL W515L. Consistent with Rela-knockout, Myd88-deficiency also diminished pathogenic WBC and monocyte count upsurgence, inhibited the accumulation of BM CD45.2 myeloid cells, and reduced spleen weights. The rapid lethality governed by mutant MPL was also delayed upon Myd88 loss, consistent with reduced pathology in spleen and BM sections. While Myd88 and Rela activate NFκB, miR-146a is repressive. We performed further knock-out of MIR146A using PDX models and transplanted MIR146A-KO CD34+ cells into NSGS mice and observed enhanced leukemic engraftment, increased spleen weights, earlier lethality, and evidence of BM sclerosis or fibrosis across both MF PDXs. Importantly, these disease parameters remained unchanged in a NBM PDX following MIR146A-KO. Lastly, in our WashU Cohort of RNA-sequencing performed on 90 CD34+ samples encompassing the spectrum of MPN and secondary acute myeloid leukemia (AML) with NBM controls, we observed significant upregulation of IRAK4 and IL1B in MF and sAML compared to NBM, and their significant expression correlation in MPN. We then pharmacologically inhibited IRAK4 using a small molecule, CA-4948, which is currently under evaluation in phase 1/2a trials of relapsed/refractory AML/myelodysplastic syndrome. In PDX experiments, CA-4948 treatment effectively reduced leukemic engraftment and spleen weights of two MF models without inducing toxicity in a NBM PDX. Through qRT-PCR and mass cytometry, we demonstrate that CA-4948 treatment dampened inflammatory cytokine production induced by IL-1β in primary MPN CD14+ monocytes. Overall, we demonstrate that targeting mediators including Rela, Myd88, and IRAK4 specifically alleviated MPN disease burden without toxicity to healthy tissue and further establish CA-4948 as a promising therapeutic avenue for the treatment of MPN.

Suppression of KRAS and CBL Mutations and Hematological Improvement By Lenzilumab and Azacitidine Treatment in Proliferative Chronic Myelomonocytic Leukemia

Introduction: Mutations in RAS-pathway genes ( NRAS, KRAS, CBL) are amongst the most common somatic mutations in cancer and historically resistant to most therapies. Targeted therapies that impact the RAS-pathway constitute a major unmet need in oncology. The proliferative form of chronic myelomonocytic leukemia (CMML) is commonly associated with RAS-pathway mutations and has a high propensity to develop into acute myeloid leukemia. In pre-clinical models, Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ), a proprietary Humaneered ® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF, resulted in a reduction of colony numbers and viability of CMML cells, with the greatest sensitivity in cells possessing RAS-pathway ( NRAS/ KRAS/ CBL) mutations, suggesting benefit in the targeted treatment of CMML. The PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M) trial assesses the efficacy of LENZ, in addition to azacytidine (AZA), in CMML subjects with RAS-pathway mutations and high dose sodium ascorbate (ASC) and AZA in CMML subjects without RAS-pathway mutations. Interim data from 11 subjects with RAS-pathway ( NRAS/ KRAS/ CBL) mutations in PREACH-M receiving LENZ/AZA demonstrate reductions in circulating GM-CSF and CRP with 8 subjects achieving complete response or optimal marrow response. This report describes improvements in variant allele frequencies (VAF) for RAS-pathway mutations and hematologic improvements associated with LENZ/AZA treatment in CMML. Methods: PREACH-M is a Phase 2/3 non-randomized, open-label trial in 72 subjects, aged at least 18 years, with newly diagnosed CMML based on the WHO 2016 criteria; and RAS-pathway mutations at a variant allele frequency ≥3%. Subjects received 24 cycles (every 28 days) of AZA (SC; 75 mg/m 2 for 7 days) and LENZ (IV; 552 mg; d1 & d15 of cycle 1 and d1 only for all subsequent cycles). Subjects without RAS-pathway mutations received the same AZA regimen and sodium ascorbate (IV; 30g for 7 days (15g for 1st dose only; 30g thereafter if no evidence of tumor lysis syndrome); PO; 1.1g on all other days). VAF was determined from bone marrow mononuclear cells using a 41-myeloid panel using Illumina Hi-Seq with a depth of 1000x performed on bone marrow aspirates obtained at baseline and Day 1 of treatment cycles 4, 7, and 12. Results: As of July 2023, 11 subjects completed at least 3 months of LENZ/AZA treatments and follow-up sequencing data were available for 10 subjects. Five of 10 subjects showed >10% decrease in VAF for at least one mutation detected at baseline, including 5 decreased alleles in the RAS-pathway ( KRAS, CBL) out of 22 total VAF mutation responses. CBL mutations showed the largest change with a decrease in >50% VAF in three subjects. Decreases in RAS-pathway clones by LENZ/AZA were durable at 6 months. No decreases in VAF for any mutations were observed in 2 evaluable subjects following ASC/AZA treatment. Following 3 months LENZ/AZA treatment, blood monocyte count improved from a baseline of 11.0 ± 6.0 x 10 9/L to 2.0 ± 1.0 x 10 9/L (p=0.030). Blast differential improved from 10.5 ± 5.0% to 4.0 ± 3.5% (p=0.038). Platelet count increased from 90.0 ± 50.0 x 10 9/L to 150.0 ± 60.0 x 10 9/L (p=0.010). Statistical improvements remained durable for at least 6 (n=6) and 12 (n=3) months. Hemoglobin concentration increased 100.0 ± 22.0 g/L to 115.0 ± 10.0 g/L at 3 months (p=NS, n=11) and progressively increased to 122.0 ± 10.0 g/L at 12 months (p=0.024, n=3). Spleen length decreased from 15.0 ± 4.0 cm to 13.0 ± 4.0 cm (p=0.03) and decreased further to 11.0 ± 2.0 cm in subjects who received 12 months of treatment (n=3). No evidence of disease progression or relapse was observed in any subject. Conclusion: In 11 evaluable subjects with proliferative CMML and RAS-pathway mutations, GM-CSF neutralization with LENZ in addition to AZA standard of care, resulted in significant decreases in the proportion of KRAS and CBL mutant leukemic cells, accompanied by clinically significant hematologic improvements and a reduction in splenomegaly. Lenzilumab may have efficacy in preventing outgrowth of RAS-pathway mutations, specifically KRAS and CBL, in the context of CMML and other myeloid malignancies.

A Journey Through JAK Inhibitors for the Treatment of Myeloproliferative Diseases.

Chronic myeloproliferative neoplasms (MPN) represent a group of diseases characterised by constitutive activation of the JAK/STAT pathway in a clonal myeloid precursor. The therapeutic approach aims to treat the symptom burden (headache, itching, debilitation), splenomegaly, slow down the fibrotic proliferation in the bone marrow and reduce the risk of thrombosis/bleeding whilst avoiding leukaemic transformation. In recent years, the advent of JAK inhibitors (JAKi) has significantly broadened treatment options for these patients. In myelofibrosis, symptom control and splenomegaly reduction can improve quality of life with improved overall survival, not impacting progression into acute leukaemia. Several JAKi are available and used worldwide, and combination approaches are now being explored. In this chapter, we review the approved JAKi, highlighting its strengths, exploring potential guidelines in choosing which one to use and reasoning towards future perspectives, where the combinations of therapies seem to promise the best results.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Advances in Risk Stratification and Treatment of Polycythemia Vera and Essential Thrombocythemia.

Estimating and modifying thrombotic risk is currently the mainstay of care for patients with polycythemia vera (PV) and essential thrombocythemia (ET). In recent years, however, increased attention has shifted towards quality of life and disease modification. In this review, we discuss recent advances in risk stratification, present updated results for ruxolitinib and interferon randomized clinical trials, discuss new approaches in antiplatelet and anticoagulant treatment, and summarize early phase trials of novel agents and emerging therapeutic concepts for the treatment of PV and ET. International collaborations and novel technologies, i.e., next-generation sequencing and machine learning techniques, have demonstrated excellent abilities to improve thrombotic risk stratification in PV and ET. Updated results from ruxolitinib and interferon randomized clinical trials have confirmed excellent efficacy and safety of these agents, both as first- and second-line treatments. Early trials of novel agents (histone deacetylase inhibitors, telomerase inhibitors, lysine-specific demethylase-1 inhibitors, human double-minute 2 inhibitors, and hepcidin mimetics) have shown encouraging efficacy and safety in blood count control, reduction of splenomegaly, and alleviation of disease-related symptoms. Finally, accumulating evidence suggested that direct oral anticoagulants may be a valid therapeutic alternative to warfarin for prolonged thromboprophylaxis. International collaborations ("big data") with the help of new technologies represent an exciting new approach to analyze rare outcomes in rare diseases, especially for identifying novel prognostic biomarkers in PV and ET. Randomized clinical trials are also needed to fully elucidate whether novel agents may establish new standards of care.

Pacritinib for the treatment of patients with myelofibrosis and thrombocytopenia

Introduction : Myelofibrosis (MF) is a rare myeloproliferative neoplasm characterized by a complex symptom profile, cytopenias, splenomegaly, and potential for leukemic progression. Severe thrombocytopenia is common in patients with MF and correlates with poor prognosis; however, until recently, treatment options for these patients were limited. Pacritinib, a potent Janus kinase (JAK) 2/interleukin-1 receptor-associated kinase 1 (IRAK1) inhibitor, has demonstrated significant reduction in splenomegaly, improved symptom control, and a manageable safety profile in patients with MF regardless of the severity of thrombocytopenia. Areas covered : This review will outline the pacritinib drug profile and summarize key efficacy and safety data, focusing on the 200 mg twice daily dose from phase 2 and 3 studies that formed the basis for the recent US Food and Drug Administration approval of pacritinib in patients with MF and severe thrombocytopenia (platelet counts <50 x 109/L). Expert opinion Pacritinib, with its unique mechanism of action targeting both JAK2 and IRAK1, offers patients with MF and severe thrombocytopenia a new treatment option, providing consistent disease and symptom control. Adverse events are easily manageable. Further analyses to identify ideal patient characteristics for pacritinib and other JAK inhibitors along with studies of pacritinib combinations are warranted, including in related myeloid malignancies.

Hemophagocytic syndrome associated with leishmaniasis: case report

Secondary hemophagocytic syndrome (sHLH) is a hyperinflammatory reaction which manifests with fever, cytopenia and organ damage. possible causes of sHLH include leishmaniasis. The article describes a clinical case of sHLH in patient with visceral leishmaniasis. A female 30 years old developed hectic daily fever up to 40 °C, pancytopenia, cytolytic syndrome, hyperferritin- and hypertriglyceridemia, immunoglobulin G to capsid antigens of the Epstein-Barr virus, enlarged liver and spleen a one and a half month after returning from Spain. based on the HLH-2004 and H-Score criteria, a sHLH was diagnosed, presumably associated with the Epstein-Barr virus. Immunosuppressive treatment with dexamethasone, cyclosporin-A and etoposide was started under the HLH-2004 program. Apyrexia, reduction of splenomegaly and resolution of cytolysis were achieved. The fever resumed 20 days after the start of chemotherapy, the spleen enlarged again, and therefore a diagnostic splenectomy was performed. Morphological analysis of the removed spleen revealed leishmania. After amphotericin-B therapy, the patient recovered. Chemotherapy of sHLH led to a temporary improvement for a period sufficient to verify the diagnosis and conducting of successful treatment.

Abstract 147: Bezuclastinib is a differentiated KIT inhibitor that exhibits unique selectivity to KIT A-loop mutations, minimal brain penetration, and favorable pharmacokinetic properties in preclinical models

Abstract KIT is a well characterized oncogenic driver, mutated in up to 80% of gastro-intestinal stromal tumors (GIST). Front-line imatinib is effective in many patients but durable responses in the setting of imatinib failure are rarely achieved due to complex secondary mutations that arise in the KIT ATP-binding pocket (exons 13/14) or the activation (A)loop (exons 17/18). Second-line sunitinib is active against exon 13/14 mutations but not A-loop mutations and identifying inhibitors that target the spectrum of possible mutations without incurring off-target toxicities related to broad-spectrum kinase inhibition has been challenging. Bezuclastinib (CGT9486) is a novel type I tyrosine kinase inhibitor that was designed as a potent and selective inhibitor of KIT A-loop mutations to address this unmet medical need. Herein, we present nonclinical data on the differentiated selectivity profile of bezuclastinib compared with other KIT inhibitors. We show that bezuclastinib is a low nanomolar inhibitor of KIT A-loop mutants which uniquely avoids targeting closely related kinases with known liabilities (e.g., PDGFRA, PDGFRB, CSF1R, and KDR) versus other approved inhibitors. In vivo pharmacokinetic/pharmacodynamic (PKPD) relationships have been evaluated utilizing either HMC1.2 human mast cell leukemia tumors for KIT phosphorylation or a Ba/F3-KIT-D816V splenomegaly model. In these models, plasma exposure associated with 50% reduction in splenomegaly or KIT phosphorylation was observed at 3-10 mg/kg/day corresponding to exposures 1.5-2.2 µg∙hr/mL. This correlated well with plasma exposure required to cause significant tumor growth inhibition in a GIST patient-derived xenograft model harboring an A-loop mutation (~1.3 µg∙hr/mL). Maximal efficacy in the PKPD models and tumor regression in GIST disease models was observed in exposure ranges of 19-36 or 11-44 µg∙hr/mL, respectively, which corresponds to clinically reached exposures in a recent GIST combination study of bezuclastinib with sunitinib. Importantly, in nonclinical studies, we also show that bezuclastinib has minimal brain penetration, a preferred feature of an anti-KIT molecule due to CNS-related adverse events observed in this targeted class. We present a tissue distribution study which demonstrates a brain:plasma ratio &amp;lt;0.1 following a 3-day administration at a dose that correlates with clinical exposure. This attractive selectivity and nonclinical safety profile, combined with early clinical data in advanced solid tumors, supports the potential for a best-in-class KIT inhibitor. Bezuclastinib is currently under clinical investigation in advanced SM (Phase 2, Apex NCT04996875), non-advanced SM (Phase 2, Summit), and imatinib-resistant GIST (Phase 3, Peak). Citation Format: Anna Guarnieri, Karyn Bouhana, LouAnn Cable, Rob Rieger, Leyla Haygood, John Robinson, Shannon Winski. Bezuclastinib is a differentiated KIT inhibitor that exhibits unique selectivity to KIT A-loop mutations, minimal brain penetration, and favorable pharmacokinetic properties in preclinical models [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 147.

A Phase 1/2 Study of Single Agent Tagraxofusp, a First-in-Class CD123-Targeted Therapy, in Patients with Myelofibrosis That Is Relapsed/Refractory Following JAK Inhibitor Therapy

A Phase 1/2 Study of Single Agent Tagraxofusp, a First-in-Class CD123-Targeted Therapy, in Patients with Myelofibrosis That Is Relapsed/Refractory Following JAK Inhibitor Therapy

Wandering splenomegaly reduction after splenopexy.

Introduction and importanceA wandering spleen is characterized by excessive splenic mobility due to the laxity of its ligaments, which leads to spleen migration and its long mobile vascular pedicle is liable to torsion. The purpose of this paper was to present a wandering splenomegaly reduction after splenic detorsion and splenopexy.Case presentationA 14-year-old boy presented a symptomatic visible pelvic wandering splenomegaly with torsion of the splenic pedicle. He was submitted to laparotomy, detorsion of the spleen, and splenopexy to the left diaphragm and surrounded peritoneum. The patient had an uneventful follow-up and was discharged from the hospital on the second postoperative day. All laboratory exams went to normal. The size of the spleen reduced from the 22 × 16 × 13 cm before the treatment to 14 × 12 × 10 cm after the surgical procedure.Clinical discussionThe most relevant aspect of this communication is the reduction of the huge splenomegaly to a normal size spleen after detorsion of the splenic vessels and splenopexy to the left diaphragm. Reduction of the splenic size is well-known after distal splenorenal shunt, but this is the first publication of a spontaneous splenic size reduction after treating a congestive wandering splenomegaly by detorsion of the spleen pedicle.ConclusionEctopic congestive splenomegaly due to the splenic pedicle rotation is adequately treated by splenic detorsion and splenopexy in its proper subdiaphragmatic site, which reduces the splenic size to normal.

Response To Ruxolitinib In Patients With Intermediate-1, Intermediate-2 and High-Risk Myelofibrosis: Interim Results Of The UK Robust Trial

BackgroundRuxolitinib is a selective small molecule inhibitor of Janus kinase 1 (JAK1) and 2 (JAK2). Ruxolitinib suppresses the activity of the JAK/STAT pathway, and thus targets the underlying pathogenic cause of myelofibrosis (MF). The efficacy and safety of ruxolitinib for the treatment of MF has been demonstrated in two pivotal phase III clinical trials, COMFORT-I and COMFORT-II. Ruxolitinib demonstrated a statistically significant reduction in splenomegaly compared with placebo (in COMFORT-I) and compared with best supportive care (in COMFORT-II) and has been approved for the treatment of patients with primary myelofibrosis (PMF), post polycythaemia myelofibrosis (PPV MF) or post-essential thrombocythaemia myelofibrosis (PET-MF) based on the results of these studies. Both pivotal studies involved patients with intermediate-2 or high-risk disease according to International Prognostic Scoring System criteria. However, a number of myelofibrosis patients with intermediate-1 risk disease have significant constitutional symptoms or pain relating to splenomegaly. The ROBUST trial, a UK open label, multicentre exploratory phase 2 study of ruxolitinib for patients with PMF, PPV MF or PET-MF further investigates the response to ruxolitinib in patients with MF and includes patients with intermediate-1 risk disease in addition to patients with intermediate-2 and high-risk disease. MethodsThis open-label multicentre UK study enrolled adult patients diagnosed with PMF, PPV MF or PET-MF (World Health Organization criteria) with or without prior therapy and who had intermediate-1, intermediate-2 or high-risk disease according to the International Working Group for Myelofibrosis Research & Treatment criteria. Patients received ruxolitinib at a dose of 15 or 20 mg twice daily (according to baseline platelet counts) and continued treatment until disease progression, unacceptable toxicity, death, or withdrawal from the study. Spleen size was assessed at baseline and at 4-week intervals by palpation, and symptoms were assessed by patients using the Myelofibrosis Symptom Assessment Form (MF-SAF) at baseline and weeks 4, 12 and 24. This interim analysis presents data at 24 weeks. ResultsA total of 48 patients (risk classification: intermediate-1, n = 20; intermediate-2, n = 11; high, n = 17) were included in the study. Mean ± SD age was 69.1 ± 10.4 years and 43.8% of patients were female. Most patients had an ECOG performance status at baseline of 0 (52.1%) or 1 (35.4%). Mean spleen length at baseline was 13.9 ± 6.9 cm and mean ± SD MF-SAF total symptom score (TSS) was 16.4 ± 11.5. A ≥50% decrease in spleen length from baseline at any time was achieved in 27/48 (56.3%) patients overall, and was similar across all risk groups. At week 24 a decrease in mean spleen length from baseline of 6.4 ± 5.2 was observed overall and was similar across the three risk groups (Table). A mean ± SD improvement in TSS at 24 weeks of 9.1 ± 11.3 was observed overall and improvements were similar in patients with intermediate-1 risk and with high-risk disease (Table). ConclusionsThese results indicate that in patients with MF, ruxolitinib provides marked and sustained reductions in splenomegaly and disease-related symptoms. Symptom and spleen responses were observed across risk categories, including intermediate-1 patients, providing further evidence of the clinical benefit of ruxolitinib in patients with MF. Missing data will be updated prior to the ASH2013 meeting, allowing more robust comparison across risk groups for the different endpoints. Disclosures:Harrison:S Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Clark:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Chacko:Amgen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Knapper:Novartis: Funding for travel to overseas conference Other, Honoraria. Milojkovic:BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria. Farquharson:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ali:Novartis: Honoraria. Andrews:Novartis Pharmaceuticals UK Limited: Employment, Equity Ownership. Graham:Novartis Pharmaceuticals UK Limited: Employment. Mead:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Real-World Outcomes of Ruxolitinib for Polycythemia Vera

BackgroundRuxolitinib (RUX) is a JAK1/JAK2 inhibitor approved for the treatment of hydroxyurea (HU)-resistant or intolerant patients with polycythemia vera (PV). Approval followed the results of the phase III RESPONSE trial, which demonstrated significant benefits of ruxolitinib in this population, including decreased phlebotomy requirements, reduction in splenomegaly, and amelioration of symptom burden. Although the results from the RESPONSE trial are positive in this selected patient population, no follow-up investigation has evaluated the real-world outcomes of patients with PV treated with RUX.MethodsWe conducted a retrospective case series of patients at a single-center tertiary-care institution who were prescribed RUX for the indication of PV between January 1st, 2013 and January 1st, 2017. Inclusion criteria included: 18 years of age or older, follow up at the study institution, and a diagnosis of PV without progression to myelofibrosis (MF) or other hematologic malignancy at time of initiation of RUX. Primary outcome measures included phlebotomy requirements, change in spleen size, and symptom burden (all assessed at 32 weeks after initiation of RUX), as well as the incidence of disease progression, and adverse events including infections, thromboses and cytopenias.ResultsBaseline disease characteristics and history are summarized in Table 1. Twenty-five (16 men and 9 women) eligible patients were identified. The average age was 65.8 years (range, 51-89 years) at initiation of RUX. Patients started RUX, on average, 9.2 years after their diagnosis. Fourteen patients (56%) had follow up at or beyond 32 weeks of initiation (4 were lost to follow up, 3 started treatment before 32-week data could be captured and 4 discontinued RUX).Primary outcomes relating to burden of disease are summarized in Table 2. Seven (50%) patients with 32-week follow-up data achieved phlebotomy freedom and four (29%) patients achieved a reduction in palpable splenomegaly of at least 50% (6 remained non-palpable, and the remaining 4 patients experienced reductions in palpable splenomegaly of less than 50%.Four (16%) of 25 patients were deemed intolerant of RUX. Intolerance was reported as headache (2 patients), weight gain (1 patient), and dizziness/diarrhea (1 patient). The average time to discontinuation of the medication was 12.6 weeks. Excluding the 4 patients who discontinued RUX, patients were on the medication for an average of 70.4 weeks (range, 8.1-182 weeks). All patients who continued RUX past 32 weeks remained on the drug at their last known follow-up visit.Three (13%) of 23 patients had leukopenia while on RUX, of which only 1 (4.3%) was grade III. Seven (30%) of 23 patients had thrombocytopenia while on RUX, of which only 1 (4.3%) was grade III. Eight (32%) of patients reported headaches while on RUX, 7 (28%) reported dizziness and 7 (28%) reported diarrhea. All reported adverse events were grade I/II. Two patients progressed to MF (on average 40.7 weeks after starting RUX). None progressed to AML or MDS. One patient, who had a previous thrombotic event, had a transient ischemic attack (grade I) while receiving RUX. No patient developed a secondary malignancy during the study period. Five (20%) patients had documented infections. Two were grade II (herpes recurrence and UTI), and one grade III (colitis), which were managed in clinic or the emergency department. Two infections, both pneumonia (grade III and V), required hospitalization.One of these patients passed away from complications of a suspected lobar pneumonia 174.2 weeks after starting RUX. The patient, who had JAK2V617F-negative/exon 12-positive disease, presented with fever and cough, and was managed in the intensive care unit with broad-spectrum antibiotics before transitioning to hospice care.ConclusionsThis is the first study to evaluate the real-world effects of RUX for the treatment of PV. Patients in our case series, most of whom had failed therapy with HU, experienced an overall reduction in phlebotomy requirement, splenomegaly, and symptom burden after the initiation of RUX. The intolerance rate to RUX in this case series was comparable to, or lower than, the reported rates for HU. Although over half of patients reported an adverse effect of RUX, less than 20% discontinued the drug for this reason. The results of this case series are largely congruous with the positive results of the original RESPONSE trial. [Display omitted] DisclosuresMascarenhas:Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding; Merck: Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Incyte: Other: Clinical Trial Steering Committee , Research Funding.

Ruxolitinib-Induced Defects in DNA Repair Cause Sensitivity to PARP Inhibitors in Myeloproliferative Neoplasms

Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), which are associated with mutations in JAK2, CALR and MPL genes. Current treatment options for Ph- MPNs include cytoreductive therapy with hydroxyurea, and the JAK2 kinase inhibitors (e.g., ruxolitinib), which produce durable reductions in splenomegaly and improvement of symptoms and probably of survival, but do not eliminate the disease-initiating cell population. All three disease “driver” mutations, [JAK2(V617F), CALR(del52), and MPL(W515L)] have been detected not only in mature MPN cells, but also in MPN stem cells, and therefore these cells must be eliminated to eradicate the disease. Since JAK2 kinase inhibitors did not eliminate the disease-initiating population novel therapeutic approaches are needed. We show here that cell lines expressing JAK2(V617F), MPL(W515L) or CALR(del52) accumulated reactive oxygen species-induced lethal DNA double-strand breaks (DSBs), which were efficiently repaired thus preventing MPN cell death. Inhibition of JAK2 kinase by ruxolitinib caused downregulation of BRCA1, RAD51 and LIG4, the key proteins in two major DSB repair mechanisms, BRCA-mediated homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (NHEJ). We recently reported (Nieborowska-Skorska et al., J.Clin.Invest., 2017) that NHEJ-deficient acute myeloid leukemia (AML) quiescent stem cells and HR/NHEJ-deficient AML proliefrating stem and progenitor cells were extremely sensitive to synthetic lethality triggered by poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673 (talazoparib). Therefore, we hypothesized that PARP inhibitors should be very effective against ruxolitinib-treated HR/NHEJ-deficient MPN quiescent and proliferating cells. Olaparib, when combined with ruxolitinib caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of samples. Importantly, combination of BMN673, ruxolitinib and hydroxyurea was highly effective in vivo against JAK2(V617F)-positive murine MPN-like disease and also against pre-selected JAK2(V617F), CALR(del52), and MPL(W515L)-positive primary MPN xenografts. However, MPN samples from individual patients displayed high degree of variability in the sensitivity to JAK2 inhibitor combined with PARP inhibitor and/or hydroxyurea. The differences between susceptibility of individual MPNs to these drugs may depend on additional mutations accompanying the “driver” mutations (e.g., TET2 mutation) and on homo/heterozygosity of the “driver” mutation allele. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized pre-selected MPN cells to synthetic lethality triggered by PARP inhibitors. DisclosuresKoschmieder:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Other: Clinical Trial participation. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau.

Primary Analysis of JUMP, a Phase 3b, Expanded-Access Study Evaluating the Safety and Efficacy of Ruxolitinib in Patients with Myelofibrosis (N = 2233)

BACKGROUND: Ruxolitinib is a potent JAK1/JAK2 inhibitor that proved superior to placebo and best available therapy in the phase 3 COMFORT studies in patients (pts) with intermediate (Int)-2- or high-risk myelofibrosis (MF). In these studies, ruxolitinib led to improvements in splenomegaly and MF-related symptoms as well as prolonged overall survival. JUMP is a large, phase 3b, expanded-access trial designed to assess the safety and efficacy of ruxolitinib in pts with MF who do not have access to ruxolitinib outside a clinical trial. Here, we report findings from the primary analysis of JUMP, which includes 2233 pts in 26 countries who received ruxolitinib treatment for ≥ 2 years. METHODS: Eligible pts had Int-2- or high-risk MF, with or without splenomegaly, or Int-1-risk MF with a palpable spleen (≥ 5 cm). The starting dose of ruxolitinib was based on platelet (PLT) counts at baseline (5 mg twice daily [bid; ≥ 50 to 200 × 109/L]) and could be titrated during treatment. The primary endpoint was assessment of safety and tolerability of ruxolitinib. Additional analyses included changes in palpable spleen length and symptom scores. Pts were treated for up to 24 months after the last patient9s first visit (23 December 2014), unless discontinuation criteria were met. RESULTS: This analysis includes 2233 pts (primary MF, 59.4% [n = 1326]). At baseline, median age was 67 y (range, 18-89 y), and 59.7% were aged ≥ 65 y; 54.5% were male; median palpable spleen length was 12 cm below the costal margin; median time since diagnosis was 25.8 months. Median hemoglobin (Hb) was 106 g/L (Hb Median exposure was 12.4 months; the mean average daily dose of ruxolitinib was 28.7 mg. 67.4% of pts had dose modifications, and 27.2% had a dose interruption. The most common grade (G) 3/4 hematologic AEs were anemia (34.8%), thrombocytopenia (16.5%), and neutropenia (4.6%), which led to discontinuation in 1.3%, 2.8%, and 0.1% of pts, respectively. Nonhematologic AEs were primarily G1/2, and the most common (≥ 10%) were pyrexia (16.0%; G3/4, 2.4%), asthenia (15.4%; G3/4, 2.2%), diarrhea (12.5%; G3/4, 1.1%), and fatigue (10.0%; G3/4, 1.0%). Rates of other G3/4 AEs were low (≤ 2%), except pneumonia (4.7%), which led to discontinuation in 10 pts (0.5%). Rates of infections were generally low; all-grade infections in ≥ 5% of pts included pneumonia (7.3%), urinary tract infection (5.9%), herpes zoster (5.2%), and nasopharyngitis (5.2%). Tuberculosis was reported in 5 pts (0.2%; G3/4, At wk 24 and 48, 56.5% (874/1546) and 61.4% (656/1068) of pts with baseline splenomegaly achieved a spleen response (ie, a ≥ 50% reduction from baseline in palpable spleen length); 23.3% (360/1546) and 18.9% (202/1068) had 25% to 50% reductions at wk 24 and 48. Most pts (70.7% [1470/2079]) experienced a ≥ 50% reduction at any time (Figure); the median time to response was 5.8 wk. Pts also experienced substantial improvements in symptoms. A large proportion of pts achieved a response (ie, a clinically significant improvement) on the FACT-Lym TS at wk 24 (53.7% [753/1403]) and wk 48 (53.7% [503/937]). Similar results were seen on the FACIT-Fatigue (wk 24, 49.1% [709/1443]; wk 48, 45.6% [434/952]). CONCLUSIONS: This was the primary analysis of JUMP, the largest study of pts with MF treated with ruxolitinib. Overall, ruxolitinib was effective in pts with MF, leading to reductions in splenomegaly and symptoms in most pts. Consistent with findings from previous studies, anemia and thrombocytopenia were the most common AEs but rarely led to discontinuation. Findings from this analysis, which included pts with Int-1-risk disease, those with low platelet counts (≥ 50 to Disclosures Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Alexion: Honoraria; Gilead: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Foltz: Pfizer: Consultancy; Incyte: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Palumbo: Novartis: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Jannsen: Membership on an entity9s Board of Directors or advisory committees; Teva: Speakers Bureau; Hospira: Membership on an entity9s Board of Directors or advisory committees. Le Coutre: Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria; Novartis: Honoraria. Zaritskey: Janssen: Consultancy; Novartis: Consultancy, Speakers Bureau. Tavares: Novartis: Consultancy. Griesshammer: Gilead: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Baxalta: Consultancy, Honoraria, Speakers Bureau; AOP Orphan: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Research Funding. Bouard: Novartis Pharma AG: Employment. Perez Ronco: Novartis Pharma AG: Employment. Tiwari: Novartis Healthcare Pvt Ltd: Employment. Vannucchi: Novartis: Honoraria, Speakers Bureau; Shire: Speakers Bureau.

Impact of Ruxolitinib-Response Feature on Outcome in Myelofibrosis: A Multicentre Study on 356 Patients

Introduction.Ruxolitinib (RUX) is a JAK1/2 inhibitor able to control myelofibrosis (MF)-related splenomegaly and symptoms and improve overall-survival (OS). It is well known that long-term RUX-therapy at optimal doses contributes to clinical benefits, but impact of RUX-response feature on outcome is still relatively unknown.Aims. To report the impact of response feature on clinical outcome of 356 patients (pts) with WHO-defined MF, INT-2 or high IPSS risk, treated with RUX.Methods. A clinical database was created in 20 Italian Hematology Centres. Retrospective data on 356 MF pts treated with RUX according to prescribing information from Jan 2011 to Jun 2016 were collected. Response was defined as either one of the following at 3 (3M) and 6 (6M) months after RUX-start: reduction of splenomegaly 35% or reduction of MPN-SAF total symptom score-TSS 50% or ≥2.0 g/dL increase in hemoglobin (Hb) level. Pts were classified into four group of response: a) pts with stable response at 3M and 6M (sR); b) pts with stable non-response at 3M and 6M (sNR); c) pts with only early response at 3M (oER); pts with only late response at 6M (oLR). Survival was calculated from the start of RUX. Event-free survival (EFS) included progression to acute leukemia (AL), death and RUX discontinuation. In group comparison, the landmark approach was used at month 6 to eliminate guarantee-time bias.Results. A total of 356 (54% primary, 46% secondary to polycythemia or thrombocythemia) MF-patients were analyzed. Median age was 67 years (range 26-89) and 61% pts had a palpable splenomegaly at > 10 cm below left costal margin with a median spleen length of 11 cm. Median value of Hb and MPN-TSS were 11.1 g/dL and 20%, respectively. Median time from diagnosis to RUX-start was 16 months and median duration of RUX-therapy was 23 months. After a median of 27 months of follow-up from RUX-start, 244 patients (69%) were still receiving RUX-therapy, with 3y OS of 75% (70 deaths) and 3y EFS of 57% (16 deaths, 16 progressions to AL, 84 RUX discontinuation). Among the four group of response, we observed 229 sR, 74 sNR, 26 oER and 27 oLR. Using a 6-month landmark approach, 3y OS according to different group of response was 74%, 58%, 39%, and 70% for sR, sNR, oER and oLR, respectively. Using a Cox model adjusted for age and gender, patients oER showed a significantly worse OS respect to sR or oLR patients (HR=2.11, p=0.025) (Figure 1).Discussion. In MF-patients a RUX-response at 6M (both stable response and only late response) seems to be associated with a clinical benefit in terms of OS. Despite the cohort of pts will be certainly further enriched, these preliminary data suggest the need to continue RUX-therapy for at least 6 months. [Display omitted] DisclosuresBonifacio:Incyte: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Tiribelli:Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Abruzzese:Novartis: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; BMS: Consultancy. Cavazzini:Incyte: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Crugnola:BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Vitolo:Mundipharma: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cuneo:Gilead: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Breccia:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Incyte: Consultancy.

Ruxolitinib Inhibits Toll-like Receptor or GM-CSF, but Not TREM-1 Mediated Neutrophil Activation

Introduction: Ruxolitinib (RUX) is the first Janus kinase (JAK)1/2 inhibitor approved for the treatment of myelofibrosis (MF) or polycytemia vera (PV), as the pathogenesis of these myeloproliferative neoplasms is linked to a dysregulation of JAK signaling. In clinical trials, RUX demonstrates a profound reduction of splenomegaly and constitutive symptoms along with an improved quality of life. The most frequent adverse events of RUX include anemia and thrombocytopenia. However, beyond this, severe infectious complications have been reported in association with RUX treatment, such as C. neoformans pneumonia, toxoplasmosis retinitis and hepatitis B virus reactivation, indicating immunosuppressive effects of this drug. On the cellular level, RUX has yet been characterized as a negative regulator for NK- and T-cell functions, possibly accounting for the side effects observed. In our present work, we focused on the impact of RUX on polymorphonuclear neutrophil (PMN) activation as important regulators of the innate immune response.Methods: Purified PMN from healthy human donors were isolated by dextran sedimentation and HistopaqueÒ centrifugation and pretreated with RUX. PMN were activated with anti-TREM-1 agonistic antibody (6B1), isotope matched control antibody (4C9) (both own production), lipopolysaccharide (LPS), zymosan or GM-CSF and effector functions like phagocytosis by fluorochrome labeled polystyrene beads, L-selectin (CD62L) shedding, oxidative burst and IL-8 secretion were analyzed. In addition, blood samples of patients under RUX treatment and samples of age matched healthy volunteers were stimulated either with GM-CSF, LPS or zymosan. Afterwards functionality of PMN was analyzed by determination of oxidative burst, phagocytosis, L-selectin shedding and degranulation.Results: Ligation of TREM-1 (by specific antibody), TLR2/6 (by zymosan), TLR4 (by LPS) or GM-CSF-R induced a strong activation of PMN derived from healthy human donors, as shown by initiation of oxidative burst, increased phagocytic activity as well as CD62L shedding and release of IL-8. The additional presence of RUX revealed an impaired phagocytosis, CD62L shedding and IL-8 production after activation with the TLR agonists (LPS, zymosan) or GM-CSF, whereas no profound impact of pretreatment was observed on TREM-1 mediated effector functions. These results suggest that the inhibition of JAK signaling affects TLR and GM-CSF mediated PMN activation, while this particular pathway appears functionally irrelevant for TREM-1 mediated PMN activation. In line with these results, we observed impaired PMN functions in patients receiving RUX treatment compared to healthy controls with the most impressive impact on the oxidative burst and phagocytosis after LPS or zymosan stimulation.Conclusion: These data provide deeper insights on the impact of RUX as an immunosuppressant of innate immunity on the cellular level adding the selective inhibition of distinct PMN activating receptor pathways. The observation of impaired effector functions of PMN after RUX pretreatment in vitro as well as in vivo may help to explain the severe infectious complications accompanying MF or PV. DisclosuresKindler:Novartis: Membership on an entity's Board of Directors or advisory committees. Radsak:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Gilead: Other: Travel support; Acceleron: Research Funding.

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of β-thalassaemia.

SummaryBeta‐thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co‐receptor for the bone morphogenetic protein (BMP)‐mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in β‐thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β‐thalassaemia mice (Hbbth3/+). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of β‐thalassaemia and related disorders.

A drug targeting 5-lipoxygenase enhances the activity of a JAK2 inhibitor in CD34+ bone marrow cells from patients with JAK2V617F-positive polycythemia vera in vitro.

Janus kinase 2 (JAK2) inhibitors, the first targeted treatments for myeloproliferative neoplasms (MPNs), provide substantial benefits, including a marked reduction in splenomegaly and MPN-associated symptoms. However, these drugs rarely induce molecular remission in patients with MPNs. Zileuton, a 5-lipoxygenase (5-LO) inhibitor, has been demonstrated to selectively deplete hematopoietic stem cells (HSCs) expressing a JAK2 point mutation (JAK2V617F) in mouse models of JAK2V617F-induced polycythemia vera (PV). To determine the potential activity of 5-LO inhibitors in combination with JAK inhibitors against human PV HSCs, the present study first analyzed 5-LO expression in CD34+ bone marrow cells from patients with JAK2V617F-positive PV using western blotting and reverse transcription-quantitative PCR, and then examined the effect of zileuton combined with ruxolitinib on colony formation using a colony formation assay. Furthermore, cell cycle and apoptosis in CD34+ cells from patients with PV and healthy volunteers were determined by flow cytometry. In the present study, 5-LO expression was upregulated in CD34+ cells from patients with PV compared with in CD34+ cells from healthy volunteers. Higher levels of leukotriene B4, a product of the 5-LO signaling pathway, were detected in patients with PV compared with in healthy volunteers. Zileuton treatment suppressed the colony formation of CD34+ cells from patients with PV in a dose-dependent manner. Furthermore, zileuton and ruxolitinib exerted their anticancer effects by suppressing hematopoietic colony formation, inducing apoptosis and arresting the cell cycle of human CD34+ cells from patients with PV. The combination of these two drugs exerted a more beneficial effect than either agent alone. Based on these data, zileuton enhanced the antitumor activity of low-dose ruxolitinib in hematopoietic progenitor cells from patients with PV, providing conceptual validation for further clinical applications of combination treatment with ruxolitinib and zileuton for patients with PV.