A 3-Part, Phase 2 Study of Bezuclastinib (CGT9486), an Oral, Selective, and Potent KIT D816V Inhibitor, in Adult Patients with Nonadvanced Systemic Mastocytosis (NonAdvSM)

Systemic mastocytosis (SM) is characterized by mast cell infiltration of ≥ 1 extracutaneous organs and encompasses a spectrum of diagnoses that can range from a non-advanced to advanced disease (Shomali et al, 2018). There are two nonadvanced variants of SM: indolent systemic mastocytosis (ISM), which accounts for approximately 85% and smoldering systemic mastocytosis (SSM), which includes about 5% of the general SM population (Cohen et al, 2014, Jennings et al, 2014). ISM is characterized by 0 or 1 B-findings and SSM by 2 or more B findings and absence of organ damage or an associated hematologic neoplasm (Gotlib et al, 2018). There are currently no approved therapies to treat the underlying disease of ISM or SSM. Although anti-mediator therapies (e.g. anti-H1 and H2 antihistamines, leukotriene receptor antagonists, cromolyn sodium, corticosteroids) are used to control symptoms such as anaphylaxis, GI intolerance, and flushing to improve quality of life, their effectiveness and tolerability are variable. Many patients experience a persistently high symptom burden despite maximized anti-mediator therapies. Because the molecular pathogenesis of SM is driven by KIT D816V mutations in 95% of patients (Garcia-Montero et al, 2006, Jara-Acevedo et al, 2015, Vaes et al, 2017), other agents targeting this mutated kinase have been used to treat the spectrum of SM variants; however, toxicities such as cognitive impairment, GI effects, intracranial hemorrhage, and edema may limit dosing and thus efficacy. In addition to targeting KIT D816V, bezuclastinib was designed to avoid other closely related kinases with known liabilities, such as PDGFRα, PDGFRβ, wild-type KIT, VEGFR2 (KDR), and CSF1R (FMS). Furthermore, bezuclastinib has demonstrated minimal brain penetration and no CNS toxicities have been identified in preclinical studies. Preliminary clinical activity with bezuclastinib has been observed in patients with advanced solid tumors or locally advanced, unresectable, or metastatic gastrointestinal stromal tumor (GIST). A reduction in KIT exon 17 mutational burden was observed in patients treated with bezuclastinib. This reduction was temporally associated with a reduction in tumor burden supporting bezuclastinib as an active therapy in KIT-driven diseases (Wagner et al, 2018). This is a multi-center, Phase 2, double blind, placebo-controlled, 3-part clinical study to evaluate the safety, efficacy, and biomarker correlates (e.g. bone marrow mast cell percentage, serum tryptase level, and KIT D816V mutation burden) of the KIT inhibitor bezuclastinib in patients with ISM and SSM. This study will enroll patients with SSM and moderate-to-severe ISM who have inadequate control of their symptoms despite at least 2 anti-mediator treatments. Part 1 of the study is intended to determine the recommended dose of bezuclastinib in Part 2. Subjects will be randomized to placebo or 1 of 3 doses of bezuclastinib which will be administered in combination with a baseline regimen of best supportive care (BSC). Part 2 will evaluate the efficacy of bezuclastinib at the selected dose as compared with placebo. Efficacy will be measured by the reduction of symptom burden as assessed by the mastocytosis activity score (MAS), a disease specific patient reported outcome tool (Siebenhaar et al, 2018). In Part 3, patients who have completed treatment in Part 1 or Part 2 of the study, including those initially randomized to placebo, may participate in a long-term extension and receive open-label bezuclastinib in combination with BSC. The study will enroll approximately 138 subjects. Data from this study will support further development of bezuclastinib in SM. Disclosures Gotlib: Cogent Biosciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair for the Eligibility and Central Response Review Committee, Research Funding; PharmaEssentia: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kartos: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Allakos: Consultancy; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Deininger: Novartis: Consultancy, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees. DeAngelo: Incyte: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Servier: Consultancy; Takeda: Consultancy; Abbvie: Research Funding; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Payumo: Cogent Biosciences, Inc: Current Employment. Mensing: Cogent Biosciences, Inc.: Current Employment. Jolin: Cogent Biosciences: Current Employment. Sachs: Cogent Biosciences: Current Employment. George: Bristol Meyers Squibb: Consultancy; Blueprint Medicines: Consultancy; Celgene: Consultancy; Incyte Corporation: Consultancy. OffLabel Disclosure: study of investigational agent

Whole Genome Sequencing Identifies Non-KIT Mutations and Cytogenetic Aberrations in Systemic Mastocytosis but Has Limited Sensitivity for Detection of KIT D816V

Background: Systemic mastocytosis (SM) is a hematologic neoplasm characterized by the infiltration of clonal mast cells in the bone marrow or other extra-cutaneous organs. The clinical course varies between advanced and non-advanced (indolent and smoldering SM) forms of SM. The vast majority of patients harbor the activating D816V mutation in the KIT tyrosine kinase. Additional somatic mutations in other genes have been recognized as risk factors in SM. Cytogenetic aberrations are rarely found in SM but have been associated with advanced disease. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) have been described as an alternative to cytogenetics and targeted molecular genetic analysis in myeloid cancers. Aim: To assess the ability of WGS/WTS to detect cytogenetic aberrations and recurrent somatic mutations in SM. Methods: 120 patients (51 female, 69 male) diagnosed with SM were analyzed with WGS/WTS and results were compared with orthogonal data of KIT D816V PCR, targeted sequencing, and cytogenetics. 47 patients (39%) were diagnosed with advanced SM (1 mast cell leukemia, 3 aggressive SM, 43 SM with associated hematologic neoplasm). For WGS, 2x151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). BaseSpace's Tumor/Normal app v3 was used to call variants with Strelka Somatic Variant Caller v2.4.7 and structural variants (aberrations with >50bp in size) with Manta (v0.28.0). Genomic DNA from a mixture of multiple anonymous donors (Promega, Fitchburg, WI, USA) was used as normal. For WTS, 2x101 bp paired-end reads were produced with a median of 50 mio. reads per sample, aligned with STAR v2.5.0, and variants were called using Isaac Variant Caller v2.3.13. Results: WGS/WTS detected cytogenetic aberrations in 21% of patients: 2 patients displayed a complex aberrant karyotype, 3 balanced structural aberrations, 16 copy number alterations, and 6 copy number neutral losses of heterozygosity. Aberrations detected by chromosome banding analysis were also found by WGS in all but three patients (small clones with aberrations present in ≤20% of metaphases and <10% of interphase nuclei as determined by FISH). In contrast, WGS/WTS detected additional aberrations in 16 patients. The frequency of chromosomal aberrations detected by WGS/WTS was higher in advanced compared to non-advanced SM (34% vs. 12%, p<0.05). KIT D816V was detected by PCR in 98%, by WGS in 21% and by WTS in 46% of patients. The detection rate by WGS was significantly higher in advanced (36%) compared to non-advanced SM (12%, p<0.05) while no difference was observed for WTS (45% vs. 47%). Somatic mutations outside of KIT were analyzed within a subset of 121 genes recurrently mutated in hematologic neoplasms. 46% of patients showed non-KIT mutations with a median of 2 mutations per patient. Both frequency of non-KIT mutations as well as the median number of mutations per patient was higher in advanced (83%; n=3) compared to non-advanced SM (22%, n=1, p<0.05). Finally, we analyzed the impact of genetic aberrations on survival in our SM cohort. Patients were grouped according to the presence of chromosomal aberrations and gene mutations (non-KIT) as assessed by WGS/WTS. SM patients with both types of aberrations (n=16), one type of aberration (n=47; gene mutations only n=38; chromosomal aberrations only n=8), or no aberration but KIT D816V (n=57) showed significant differences in overall survival (p<0.05, Figure 1). Con clusions: WGS/WTS has limited sensitivity for detection of KIT D816V in SM. This finding can be explained by the low KIT D816V mutation burden typically found in bone marrow aspirates of SM patients. In line, we observed a slightly higher detection rate in advanced SM and in RNA-based WTS analysis. As WGS/WTS will be applied for the diagnostic workup of myeloid malignancies in the future and SM associated with other hematologic neoplasms may be overlooked if not specifically investigated, additional PCR-based techniques are still mandatory to rule out KIT D816V as a diagnostic criterion for SM. In contrast, WGS/WTS detects both chromosomal aberrations and additional gene mutations in patients with SM and can be used as an alternative to cytogenetics and targeted sequencing for risk assessment. In particular, the absence of genetic aberrations in WGS/WTS identifies SM patients with indolent course of the disease and favorable prognosis. Figure 1 Figure 1. Disclosures Hoermann: Novartis: Honoraria. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Preclinical Data with KIT D816V Inhibitor Bezuclastinib (CGT9486) Demonstrates High Selectivity and Minimal Brain Penetrance

The molecular pathogenesis of Systemic Mastocytosis (SM) is driven by mutations in the KIT gene, with 95% of patients having a mutation in exon 17, D816V, leading to constant proliferation of mast cells (Garcia-Montero et al, 2006; Jara-Acevedo et al, 2015; Vaes et al, 2017). Targeted therapeutics have revealed clinical activity in these patients, but toxicities such as cognitive effects, intracranial hemorrhage, hypertension, and edema may limit dosing and availability of these therapies. While the exact cause of these effects is difficult to determine, numerous closely related kinases, such as wild type PDGFRα, PDGFRβ, KIT, VEGFR2 (KDR), and CSF1R (FMS), are considered to be anti-targets, with previous evidence of their inhibition linked to observed clinical toxicities (Liu & Kurzrock, 2015; Giles et al., 2009; Jayson et al., 2005). Bezuclastinib (CGT9486) was designed to selectively inhibit KIT D816V versus these other closely related kinase anti-targets. Additionally, we demonstrate that bezuclastinib has minimal brain penetration, together with no observed CNS-related toxicities in nonclinical studies. Herein, we present results from cell-based kinase profiling assays, which demonstrate that bezuclastinib has a significant and unique selectivity to KIT D816V relative to the aforementioned kinases when tested head-to-head against other clinically relevant compounds in SM. Additionally, a similar selectivity profile was observed for a broader panel of kinases, ion channels, transporters, and enzymes, which will be presented here, including drug concentrations and target engagement achieved with recent in vivo studies. Importantly, 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 these indications. In a tissue distribution study performed in rats, bezuclastinib shows a brain:plasma ratio <0.1 following 3 day administration at 25 mg/kg, a dose that closely correlates with clinical plasma exposure. This was supported functionally by assessing neurobehavioral effects of bezuclastinib at dose levels up to 100 mg/kg in rats which showed no CNS related effects. This attractive selectivity and nonclinical safety profile, combined with early clinical data in advanced solid tumors (Trent et al, 2020), supports the potential for a best-in-class KIT inhibitor. Bezuclastinib is currently under clinical investigation in advanced SM with additional clinical studies planned in non-advanced SM and imatinib-resistant GIST. Disclosures Guarnieri: Cogent Biosciences: Current Employment. Cable: Cogent Biosciences: Current Employment. Bouhana: Cogent Biosciences: Current Employment. Sullivan: Cogent Biosciences: Current Employment. Ball: Cogent Biosciences: Current Employment. Sachs: Cogent Biosciences: Current Employment. Winski: Cogent Biosciences: Current Employment. Robinson: Cogent Biosciences: Current Employment.

CD117 As an Immunotherapeutic Target in Advanced Forms of Mastocytosis

Mastocytosis is a malignant disease resulting from oncogenic transformed mast cells. Up to 80% of malignant cells harbor a D816V mutation in the KIT-receptor (CD117), leading to constitutive kinase activation and proliferation and survival of mast cells. Advanced forms of mastocytosis (aggressive systemic mastocytosis: ASM, systemic mastocytosis with associated hematological disease: SM-AHN, mast cell leukemia: MCL) present as a therapeutic challenge. Although the recently approved poly tyrosine kinase inhibitor Midostaurin provides some improvement, the median overall survival ranges from 3.5 years (ASM) to less than six months (MCL). The reduced life expectancy is frequently due to mast cell infiltration resulting in multi organ failure. Additionally, there are patients who do not benefit from the treatment with Midostaurin (overall response 60%) or suffer from side effects, which lead to reduction or termination of therapy. Currently, the only available curative approach is conditioning poly-chemotherapy followed by allogenic stem cell transplantation (allo-HSCT). However, allo-HSCT is associated with substantial side-effects and, also due to high rates of relapse, only leads to an overall survival of 43% for ASM and 17% for MCL after three years. Thus, better therapeutic options are needed. Recently, we demonstrated that CD117 (KIT-receptor) positive human AML can be efficiently eradicated by anti-CD117 CAR T-cells in vitro and in vivo (Myburgh et al., Leukemia 2020). As mast cells, and also transformed mast cells, highly express CD117, we here tested if anti-CD117 CAR T-cells would equally efficiently eliminate this malignant cell population. We thus co-cultured various established mast cell lines (partly harboring the oncogenic driver mutation KIT D816V) with anti-CD117-CAR T-cells in a 1:1 effector to target ratio in vitro. After 24 hours of co-culturing, the tumor cells were effectively killed, and this was still observed despite increasing the effector to target ratio to 1:4. Also, within 28 days of co-culture, the longest time followed in vitro, tumor cells were controlled and did not outgrow. Increased proliferation of anti-CD117-CAR T-cells in the presence of mast cells was observed and tracked throughout the 28-day experiment. In conclusion, we demonstrate that the human mast cell lines HMC-1.1 KIT V560G, HMC-1.2 KIT V560G, KIT D816V, ROSA KIT WT, ROSA KIT D816V, LAD2 and MCPV-1 can be efficiently targeted and killed in vitro by allogeneic anti-CD117-CAR T-cells. Given that CD117 is expressed on healthy hematopoietic stem and progenitor cells (HSPCs) on a substantially lower level, there might be a therapeutic window for anti-CD117 immunotherapy in advanced forms of mastocytosis. However, as CAR T-cells are highly efficient, collateral damage on healthy HSPCs will likely need to be compensated by subsequent HSC transplantation. We are currently translating these promising in vitro immunotherapeutic settings into surrogate xenogeneic in vivo models. Disclosures Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

PEDIATRIC MASTOCYTOSIS: AN UPDATE

Mastocytosis is a rare clonal disorder, characterized by excessive proliferation and accumulation of mast cells (MC) in various organs and tissues. Cutaneous mastocytosis (CM), the most common form in children, is defined when MC infiltration is limited to the skin. Systemic mastocytosis (SM), the most common form in adults, is characterized by MC proliferation and accumulation in organs, such as bone marrow, lymph nodes, liver and spleen (1). Genetic aberrations, mainly the KIT D816V mutation, play a crucial role in the pathogenesis of mastocytosis, resulting in enhancing MC survival and subsequent accumulation in organs and tissues (2,3). CM includes 3 forms: solitary mastocytoma, maculopapular cutaneous mastocytosis (MPCM) and diffuse cutaneous mastocytosis (DCM). In the majority of children with CM, skin lesions regress spontaneously around puberty; unfortunately, in a few cases, it is not a self-limiting disease (4). Even if SM occurs occasionally, all children with mastocytosis require planned follow-up over time. Children with mastocytosis often suffer from MC mediator-related symptoms, the most common of which is itching, often triggered by rubbing the lesions. Management of pediatric mastocytosis is mainly based on strict avoidance of triggers. Treatment with H1 and H2 histamine receptor blockers on demand, and the availability of epinephrine auto-injectors for the patients to use in case of severe anaphylactic reactions are recommended.

Кожна мастоцитоза в детска възраст: клиничен случай и литературен обзор

Мастоцитозата (М) представлява хетерогенно заболяване, характеризиращо се с повишено натрупване и клонална пролиферация на мастоцити в кожата и/или различни органи. Най-общо М се класифицира в две категории: кожна мастоцитоза (СМ) и системна мастоцитоза (SМ). При деца CM е най-честата форма, но лезиите се различават според клиничната форма. Представяме клиничен случай на момче на 1 година и 4 месеца, при което на 4-месечна възраст, след тежка алергична реакция е поставена диагноза солитарен мастоцитом, потвърдена от биопсия на кожата. Мастоцитомът е забелязан от родителите 4 дни след раждането. Пациентът има повишени нива на хистамин и серумна триптаза. Генетичният маркер KIT D816V в кръвта е отрицателен. Децата често страдат от симптоми, свързани с отделянето на медиатори от мастоцитите. Могат да се появят и тежки реакции на свръхчувствителност, най-вече при пациенти с обширни кожни лезии и образуване на мехури. Оценката на педиатричния пациент с CM обикновено включва лабораторни изследвания (серумна триптаза), кожна биопсия и обстоен клиничен преглед. Определящо е също така да се прави разлика между СМ и други заболявания с кожно засягане. Лечението на CM в детска възраст се основава главно на стриктно избягване на тригерите. Наличните лекарства са перорални Н1 и/или Н2 антихистамини, перорален натриев кромогликат, фотохимиотерапия (перорална метоксипсораленова терапия и комбиниране на псорален с ултравиолетови А лъчи), мощни дермокортикоиди, калциневринови инхибитори и обучение на пациентите и техните семейства за приложение на адреналинови автоинжектори при тежки анафилактични реакции. При децата прогнозата за CM е добра и в повечето случаи кожните лезии регресират спонтанно около пубертета. Хетерогенните симптоми на различните подварианти на мастоцитозата изискват мултидисциплинарно сътрудничество и цялостна грижа.

The association of Greig syndrome and mastocytosis reveals the involvement of hedgehog pathway in advanced mastocytosis

Mastocytosis is a heterogeneous disease characterized by an abnormal accumulation of mast cells (MCs) in one or several organs. Although a somatic KIT D816V mutation is detected in ~85% of patients, attempts to demonstrate its oncogenic effect alone have repeatedly failed, suggesting that additional pathways are involved in MC transformation. From three children presenting with both Greig cephalopolysyndactyly syndrome (GCPS, MIM#175700) and congenital mastocytosis, we demonstrated the involvement of the hedgehog (Hh) pathway in mastocytosis. GCPS is an extremely rare syndrome resulting from haploinsufficiency of GLI3, the major repressor of Hh family members. From these familial cases of mastocytosis, we demonstrate that the Hh pathway is barely active in normal primary MCs and overactive in neoplastic MCs. We show that GLI3 and KIT mutations have a synergistic, tumorigenic effect on the onset of mastocytosis in a GCPS mouse model. Finally, we show that Hh inhibitors suppress neoplastic MC proliferation in vitro and extend the survival time of aggressive systemic mastocytosis (ASM) mice. This work revealed, for the first time, the involvement of Hh signaling in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in ASM, leading to the identification of new promising therapeutic targets.