Efficacy and safety of avapritinib in advanced systemic mastocytosis: Interim analysis of the phase 2 PATHFINDER trial

Advanced systemic mastocytosis (AdvSM) is a rare, KIT D816V-driven hematologic neoplasm characterized by mast cell infiltration and shortened survival. We report the results of a prespecified interim analysis of an ongoing pivotal single-arm phase 2 trial (no. NCT03580655 ) of avapritinib, a potent, selective KIT D816V inhibitor administered primarily at a once-daily starting dose of 200 mg in patients with AdvSM (n = 62). The primary endpoint was overall response rate (ORR). Secondary endpoints included mean baseline change in AdvSM-Symptom Assessment Form Total Symptom Score and quality of life, time to response, duration of response, progression-free survival, overall survival, changes in measures of disease burden and safety. The primary endpoint was successfully met (P = 1.6 × 1

Altered translation of GATA1 in Diamond-Blackfan anemia

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA),1,2 congenital asplenia,3 and T-cell leukemia.4 Yet how mutations in such ubiquitously expressed proteins result in cell-type and tissue specific defects remains a mystery.5 Here, we show that GATA1 mutations that reduce full-length protein levels of this critical hematopoietic transcription factor can cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can similarly reduce translation of GATA1 mRNA - a phenomenon that appears to result from this mRNA having a higher threshold for initiation of translation. In primary hematopoietic cells from patients with RPS19 mutations, a transcriptional signature of GATA1 target genes is globally and specifically reduced, confirming that the activity, but not the mRNA level, of GATA1 is reduced in DBA patients with ribosomal protein mutations. The defective hematopoiesis observed in DBA patients with ribosomal protein haploinsufficiency can be at least partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations in ubiquitous ribosomal proteins can result in human disease

Targeting the JAK2-V617F Mutation in Polycythemia Vera Using CRISPR/AAV6 Genome Editing

Polycythemia Vera (PV) is a chronic myeloproliferative neoplasm characterized by an overproduction of red blood cells (RBCs) and can progress to myelofibrosis and acute myeloid leukemia (AML). In ~ 95% of patients, PV is associated with the JAK2-V617F gain-of-function mutation. This mutation results in constitutive activation of the JAK/STAT signaling pathway, circumventing the normal erythropoietin-dependent regulation of erythropoiesis. Current therapies, such as hydroxyurea, interferon-alfa and ruxolitinib, primarily elicit hematologic and symptom responses, with some potential for reduction of JAK2-V617F allele burden, but are not curative. The challenge in developing definitive treatments for PV lies in the dominant nature of the JAK2-V617F mutation and the capacity of mutant cells to clonally expand. This necessitates the complete eradication or correction of all mutant cells to prevent disease recurrence. To address these limitations, we present a novel genome editing approach utilizing CRISPR-Cas9 RNP in conjunction with AAV6 delivery, aimed at precise correction of the JAK2-V617F mutation in hematopoietic stem cells (HSCs), potentially enabling autologous stem cell transplantation as a curative therapy. Our genome editing strategy employs a dual-guide CRISPR-Cas9 system designed for high-efficiency correction of the JAK2-V617F mutation. This approach utilizes two guide RNAs: one specifically targeting the V617F mutation site, and another directed at an intronic region ~100 base pairs downstream. The mutation-specific guide alone shows 51±7.9% indel formation and consequent mutation deactivation. However, the synergistic effect of the dual-guide system significantly enhances editing efficiency, resulting in ~99% knockout or deactivation of the mutant allele in affected cells. Notably, this approach preserves wild-type alleles, with only intronic indels observed in non-mutant sequences. To facilitate translational applications, we engineered two homology directed repair (HDR) AAV6 vector constructs with varying homology arm sequences: HA1-JAK2cDNA-PGK-tNGFR and HA2-JAK2cDNA-PGK-tNGFR. Both vectors integrate codon optimized cDNA of the downstream exons along with a PGK promoter-driven truncated nerve growth factor receptor (tNGFR) as a selection marker, allowing MACS purification of edited cells. When applied to HSCs, these vectors successfully edited 43.1±10.2% and 46.8±7.6% of cells, respectively, as determined by the presence of the tNGFR marker. MACS purification yielded enriched populations of edited cells, with 96.6±1.9% and 97.2±1.6% purity for the two vectors, respectively. To assess the clinical relevance of our genome editing approach, we developed an in vitro model utilizing HSCs isolated from bone marrow aspirates of PV patients, obtained through a Stanford IRB-approved protocol. This model recapitulated key pathophysiological features of PV, including accelerated erythroid differentiation, enhanced erythrocyte proliferation, and an elevated mutant allele frequency. Our editing strategy, applied to patient-derived samples without MACS purification, eliminated 94.8±3.4% of mutant alleles, with 14.9±0.3% of these alleles reverting to the wild-type sequence. Notably, this genetic correction led to functional improvements. In a 14-day erythroid differentiation culture, our approach mitigated the aberrant cellular phenotype, reducing early erythrocyte proliferation and differentiation on day 4 by 7.4±6.6 and 33.4±8.5 fold, respectively. These reductions effectively restored cellular behavior to levels comparable with healthy control HSCs. Crucially, the overall erythrocyte differentiation capacity remained intact post-editing, with 94.8±6.7% of edited patient samples maturing into RBCs as evaluated by CD34-/CD45-/CD71+/CD235a+ immunophenotyping. These findings demonstrate our editing strategy's potential to correct the mutation, normalize cellular phenotypes, and preserve hematopoietic functions. These preliminary results demonstrate an early proof of concept for developing a one-time gene therapy for PV. While further long-term studies, including serial transplantation experiments, are needed to assess the impact on clonal expansion, the integration of this genome editing strategy with emerging stem cell engraftment approaches may serve as a starting point for advancing PV treatment

Altered translation of GATA1 in Diamond-Blackfan anemia

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA)[superscript 1, 2], congenital asplenia[superscript 3] and T cell leukemia[superscript 4]. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type– and tissue-specific defects remains unknown[superscript 5]. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.National Institutes of Health (U.S.) (Grant P01 HL32262)National Institutes of Health (U.S.) (Grant U54 HG003067-09

Refined treatment response criteria for indolent systemic mastocytosis proposed by the ECNM-AIM consortium

Indolent systemic mastocytosis (ISM) has a favorable prognosis and normal life expectancy. However, many patients suffer from mast cell (MC) mediator-related symptoms, which significantly affect quality of life (QoL). Cutaneous, gastrointestinal, and neurological complaints, musculoskeletal pain, and the presence of skin lesions, anaphylaxis, and osteoporosis are the main symptoms and signs in ISM and must be assessed in all patients before and during treatment. Validated mastocytosis-specific patient-reported outcome measures (PROMs) should be used for this purpose. Serum tryptase and KIT D816V allele burden are recommended as secondary outcome parameters, noting that they do not reflect the severity of signs, symptoms, and related QoL impairment, but indirectly express MC burden. Changes from baseline of 90%, 60%, and 30% indicate complete response >90%, major response 60% to 90%, partial response 30% to 60%, and no response <30% to treatment. To conclude, we recommend the use of PROMs as primary outcome parameters to define treatment response in patients with ISM in clinical trials and in everyday clinical practice.M. C. Carter, J. J. Lyons, and D. D. Metcalfe were supported by the Division of Intramural Research, National Institutes of Allergic and Infectious Diseases, and National Institutes of Health. M. Niedoszytko was supported by the Medical University of Gdansk grant 02-0141/07/231. P. Valent was supported by the Austrian Science Fund (FWF) grant # P32470-B

Clinical impact and proposed application of molecular markers, genetic variants, and cytogenetic analysis in mast cell neoplasms: Status 2022

Mast cell neoplasms are an emerging challenge in the fields of internal medicine, allergy, immunology, dermatology, laboratory medicine, and pathology. In this review, we discuss the current standards for the diagnosis and prognostication of mast cell neoplasms with special reference to clinically relevant germline and somatic gene variants. In patients with cutaneous mastocytosis or with indolent systemic mastocytosis (SM), various KIT-activating mutations act as key molecular drivers of the disease. In adults, KIT p.D816V is by far the most prevalent driver, whereas other KIT mutants are detected in nearly 40% of children. In advanced SM, including aggressive SM, SM with an associated hematological neoplasm, and mast cell leukemia, additional somatic mutations in other genes, such as SRSF2, JAK2, RUNX1, ASXL1, or RAS, may be detected. These drivers are more frequently detected in SM with an associated hematological neoplasm, particularly in male patients. Recently, hereditary alpha-tryptasemia has been identified as a genetic trait more prevalent in SM compared with healthy controls. Moreover, hereditary alpha-tryptasemia is more frequent in patients with SM with Hymenoptera venom allergy and severe mediator-related symptoms than in patients with SM without symptoms. On the basis of this knowledge, we propose a diagnostic algorithm in which genetic markers are applied together with clinical and histopathologic criteria to establish the diagnosis and prognosis in SM

Midostaurin improves quality of life and mediator-related symptoms in advanced systemic mastocytosis

Background: Advanced systemic mastocytosis (advSM) is characterized by presence of the KIT D816V mutation and pathologic accumulation of neoplastic mast cells (MCs) in various tissues, leading to severe symptoms and organ damage (eg, cytopenias, liver dysfunction, portal hypertension, malabsorption, and weight loss). Treatment with midostaurin, an orally active multikinase/KIT inhibitor now approved for advSM in the United States and the European Union, resulted in a high rate of response accompanied by reduced MC infiltration of the bone marrow and lowered serum tryptase level. Objective: We aimed to determine whether midostaurin improves health-related quality of life (QOL) and MC mediator related symptoms in patients with advSM. Methods: In 116 patients with systemic mastocytosis (89 patients with advSM fulfilling the strict inclusion criteria of the D2201 study [ClinicalTrials.gov identifier NCT00782067]), QOL and symptom burden were assessed during treatment with midostaurin by using the 12-Item Short-Form Health Survey (SF-12) and the Memorial Symptom Assessment Scale patient reported questionnaires, respectively. MC mediator related symptoms were evaluated by using a specific physician-reported questionnaire Results: Over the first 6 cycles of treatment with midostaurin (ie, 6 months), patients experienced significant improvements in total SF-12 and Memorial Symptom Assessment Scale scores, as well as in subscores of each instrument. These improvements were durable during 36 months of follow-up. Similarly, we found substantial improvements (67%-100%) in all MC mediator related symptoms. Conclusion: QOL and MC mediator related symptoms significantly improve with midostaurin treatment in patients with advSM (ClinicalTrials.gov identifier, NCT00782067)

Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R