Small Molecule Inhibitor of Neutrophil Elastase Normalizes Myeloid Differentiation and Improves Impaired Cell Survival Triggered by Elastase Mutations In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

Abstract Abstract 386 Heterozygous mutations in the neutrophil elastase gene ELANE have been identified as the primary cause of severe congenital neutropenia (SCN) associated with recurring severe infections and evolution to acute myeloid leukemia (AML). As of today, more than 50 substitution, truncation, insertion and deletion mutations have been identified. Animal studies based on knock-in or knockout of ELANE in mice failed to produce severe neutropenia phenotype. We and others previously reported that expression of various mutants but not wild type neutrophil elastase (NE) in human but not murine cells triggers accelerated apoptosis. We also reported that expression of mutant NE (del.145-152), identified in SCN patients one of whom evolved to develop MDS/AML, in human promyelocytic tet-off HL60 cells causes both accelerated apoptosis and characteristic block of myeloid differentiation similar to that seen in bone marrow of SCN patients. Examination of the tertiary structure of NE revealed that most of the mutations leave the active site of the mutant protease intact. We identified a small molecule inhibitor of neutrophil elastase, a derivative of L-malic acid (Merck, USA), that blocked the proteolytic activity of NE by approximately 80% and was capable of restoring impaired myeloid differentiation and normalizing production of myeloid cells expressing del145-152 NE mutant. It is important to note that block of proteolytic activity of NE with the NE-SMI had no adverse effect on control human myeloid progenitor cells expressing wild type NE, thus confirming the gain-of-function effect of NE mutants. More than 20% of SCN patients with NE mutations evolve to develop AML. Molecular modeling and analysis of the tertiary structures of NE available through the Protein Database revealed that 16 different mutations identified in AML patients affect predominantly the N95 or N144 glycosylation sites or the binding pocket of the protease suggesting that altered substrate specificity of the mutant enzyme is the cause of accelerated apoptosis and block of myeloid differentiation in SCN/AML. We sought to obtain bone marrow samples from 2 unrelated SCN/AML patients both on G-CSF treatment harboring either C122Y or insPQ94. Bone marrow purified CD34+ and/or CD34-/CD33+ myeloid progenitors from the patients showed basal level of apoptosis in a range of 20–25%, which gradually increased reaching 40–50% apoptosis by 3 days of culture. Importantly, treatment of primary bone marrow-derived cells with NE-SMI substantially reduced accelerated apoptosis to near initial rate with approximately up to 2-fold reduction of apoptosis by 3 days of culture as determined by flow cytometry. Thus, our findings demonstrate that 1) small molecule inhibitor of neutrophil elastase is effective in blocking accelerated apoptosis triggered by three different NE mutations identified in SCN patients evolved to develop MDS/AML and 2) the small molecule inhibitor of NE is a promising therapeutic agent that should be considered for testing in clinical trials in SCN/AML patients. Disclosures: Dale: Amgen: Consultancy, Research Funding; Merck: Patents & Royalties, Research Support.

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Small Molecule Inhibitor of Neutrophil Elastase and Severe Congenital Neutropenia.

Blood ◽

10.1182/blood.v114.22.552.552 ◽

2009 ◽

Vol 114 (22) ◽

pp. 552-552

Author(s):

Andrew A Aprikyan ◽

Vahagn Makaryan ◽

Maxim Totrov ◽

Ruben Abagyan ◽

David C. Dale

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Small Molecule ◽

Small Molecule Inhibitor ◽

Myeloid Differentiation ◽

Cellular Model ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Compound A ◽

Molecule Inhibitor

Abstract Abstract 552 Severe congenital neutropenia (SCN) is a rare heritable hematopoietic disorder characterized by maturation arrest at the promyelocytes, recurring severe infections, and evolution to leukemia. Heterozygous mutations in the neutrophil elastase (NE or ELANE) gene (sporadic or autosomal-dominant SCN) or homozygous mutations in the HAX1 gene (autosomal-recessive SCN) are associated with similar clinical phenotype and a block of myeloid differentiation or “maturation arrest” in the marrow. We and others reported that human myeloid progenitor cells expressing mutant elastase exhibit impaired cell survival (Aprikyan et al, 2003, Massullo et al, 2005, Kollner et al, 2006, Grenda et al, 2007). The hetero- and homozygous deletion of NE as well as the knock-in of mutant NE identified in SCN/AML patient failed to produce severe neutropenia phenotype in mice. Thus, the pathomechanism of severe neutropenia remains largely unclear due to the lack of cellular or animal model of SCN with characteristic block of myeloid differentiation and accelerated apoptosis. We established a cellular model of SCN with inducible tet-regulated expression of del.145-152 NE mutant (identified in SCN/AML patients) in human promyelocytic tet-off HL60 cells. The ratio of normal/mutant NE products in these cells is approximately 1:1, which is similar to that expected in SCN patients with heterozygous NE mutation. Expression of mutant NE in the promyelocytic cells results in a characteristic block of myeloid differentiation with ∼70% decline in differentiated neutrophils which is similar to that observed in SCN, whereas induced expression of control wild type NE has no effect on myeloid differentiation. Reduced production of myeloid cells and accelerated apoptosis are also observed upon DMSO or retinoic acid induced granulocytic differentiation of the cells in response to mutant, but not wild type NE expression. Thus, this cellular model of SCN appears to closely recapitulate the human phenotype. Expression of mutant NE resulted in approximately 40% increase in total NE-specific proteolytic activity, suggesting that mutant elastase exhibits at least some proteolytic activity. To date there are more than 50 heterozygous mutations in the NE gene have been identified in pre-leukemic SCN patients. Molecular modeling of the NE tertiary structure revealed that these mutations predominantly affect the N-glycosylation sites or the binding pocket of neutrophil elastase. Importantly, the active site of the mutant protease appears to be intact, which suggests that NE-specific small molecule inhibitors may be useful in preventing accelerated apoptosis and the characteristic block of myeloid differentiation of myeloid progenitor cells. Screening this cellular model of SCN, we identified a proprietary cell-penetrant elastase-specific small molecule inhibitor (compound A, Merck, USA), which inhibits the proteolytic activity of NE by more than 80%. When treated with compound A, control cells with induced expression of wtNE exhibit normal myeloid differentiation and production of myeloid cells, similar to that in untreated cells. These data suggest that human NE is dispensable and that accelerated apoptosis and impaired myeloid differentiation in SCN is due to a gain-of-function effect of pro-apoptotic mutant elastase. Importantly, treatment of human promyelocytic cells expressing del.145-152 mutant NE with this small molecule inhibitor restores impaired production of myeloid cells and improves myeloid differentiation to near normal levels, thus neutralizing the pro-apoptotic effect of mutant NE. These data suggest that small molecule inhibitors of NE may represent a promising therapy in severe congenital neutropenia. We have examined the effect of the inhibitor on bone marrow cells from an SCN patient positive for NE mutation. At the time of bone marrow aspiration the patient was on G-CSF and the patient's freshly isolated bone marrow CD33+ progenitor cells exhibited ∼21% apoptosis, which was gradually increased reaching 43% at 3 days of culture. However, daily treatment of SCN cells with compound A preserved the cell survival rate at the initial value resulting in approximately 2-fold reduction in apoptotic cell death at 3 days of culture. These data demonstrate that the small molecule inhibitor of NE and its analogs should be considered for clinical trials in patients with SCN that is attributable to mutant NE. Disclosures: Dale: Merck: Research support; Amgen: Consultancy, Research Funding, Speaker.

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Small Molecule Inhibitor of Neutrophil Elastase Restores Impaired Production of Human Myeloid Cells Observed in Severe Congenital Neutropenia.

Blood ◽

10.1182/blood.v110.11.664.664 ◽

2007 ◽

Vol 110 (11) ◽

pp. 664-664

Author(s):

Andrew A. Aprikyan ◽

Vahagn Makaryan ◽

Qian Si ◽

Kelly Treonze ◽

Nara Markosyan ◽

...

Keyword(s):

Proteolytic Activity ◽

Small Molecule ◽

Myeloid Cells ◽

Small Molecule Inhibitor ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Compound A ◽

Maturation Arrest ◽

Molecule Inhibitor

Abstract Severe congenital neutropenia (SCN) is a rare hematopoietic disease characterized by maturation arrest at the promyelocytes, recurring severe infections, and evolution to leukemia. Heterozygous mutations in either the neutrophil elastase (NE, ELA2) gene (sporadic or autosomal-dominant SCN) or homozygous mutations in the HAX1 gene (autosomal-recessive SCN) are associated with a similar clinical phenotype and similar block of myeloid differentiation in the marrow. Most studies now indicate that the maturation arrest in SCN is due to accelerated apoptosis of myeloid progenitors triggered by the mutant gene products. The hetero and homozygous deletion of NE in mice as well as the knock-in of mutant NE identified in SCN/AML patient failed to produce severe neutropenia phenotype in mice. We established a cellular model of SCN with inducible expression of del.145–152 NE mutant in human promyelocytic tet-off HL60 cells. Ratio of normal /mutant NE products in these cells is approximately 1:1, similar to that expected in SCN patients with heterozygous NE mutation. Expression of mutant NE in promyelocytic cells resulted in a characteristic block of myeloid differentiation with ∼70% decline in differentiated neutrophils which is similar to that observed in SCN. Cell growth reduction and accelerated apoptosis were also observed in these cells in response to mutant NE expression. Thus, this SCN model appears to closely recapitulate the human phenotype. Analysis of the proteolytic activity of cells expressing mutant elastase revealed approximately 40% increase in total NE-specific activity in response to mutant NE expression compared with controls, suggesting that mutant elastase exhibits at least some proteolytic activity. To date we identified more than 40 heterozygous mutations in the NE gene in pre-leukemic SCN patients, however, the pathomechanism remains unclear. Molecular modeling of the NE tertiary structure revealed that these mutations predominantly affect the N-glycosylation sites or the binding pocket of elastase. Importantly, the active site of the mutant protease appears to be intact, which suggested that NE-specific small molecule inhibitors may be useful in preventing accelerated apoptosis and the block of myeloid differentiation of myeloid cells. Examining this SCN model, we identified a proprietary cell-permeable elastase-specific small molecule inhibitor (compound A, Merck, USA), which inhibited the proteolytic activity of the NE by more than 80%. Our studies indicate that treatment of human promyelocytic cells expressing del.145–152 mutant NE with this small molecule inhibitor restored the impaired production of myeloid cells and improved myeloid differentiation to near normal level. Importantly, the compound A did not impair the growth rate of control cells with normal NE expression. These data suggest that NE-specific small molecule inhibitor and its analogs should be considered in clinical trials in patients with SCN that is attributable to mutant NE.

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The C-Terminal Region of the G-CSF Receptor Is Required for Induction of Neutrophil Elastase Expression in Myeloid Cells: Implication for Understanding CSF3R Mutations in Acute Myeloid Leukemia Evolving from Severe Congenital Neutropenia.

Blood ◽

10.1182/blood.v108.11.1154.1154 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1154-1154

Author(s):

Fan Dong ◽

Yaling Qiu ◽

Alan D. Friedman ◽

Qingquan Liu

Keyword(s):

Acute Myeloid Leukemia ◽

Neutrophil Elastase ◽

Myeloid Leukemia ◽

Myeloid Cells ◽

Luciferase Reporter ◽

Severe Congenital Neutropenia ◽

Wild Type ◽

Congenital Neutropenia ◽

Nonsense Mutations ◽

Acute Myeloid

Abstract Severe congenital neutropenia (SCN) is characterized by early onset of bacterial infections and maturation arrest of myeloid cells at early stages of differentiation in the bone marrow. Point mutations in ELA2 encoding neutrophil elastase (NE) have been identified in 60% to 80% of patients with SCN. SCN patients are predisposed to acute myeloid leukemia (AML), which occurs in approximately 15 % of cases. With rare exceptions, leukemic cells from these patients carry mutations in CSF3R encoding the G-CSF receptor, leading to C-terminal truncation of the receptor. Notably, the nonsense mutations in CSF3R are present only in SCN/AML patients, particularly those with ELA2 mutations, but not in other types of neutropenias and de novo AML. The mechanism for the exclusive presence of the nonsense CSF3R mutations in SCN/AML is unknown. In myeloid 32D cells transfected with the wild type (WT) G-CSF receptor (32D/WT), G-CSF treatment induced the expression of NE. However, NE expression was not upregulated by G-CSF in 32D cells expressing the truncated G-CSF receptor d715, derived from an SCN patient. It has been shown that myeloid cells from patients with SCN/AML express both the wild type and the truncated G-CSF receptors. Indeed, the d715 mutant acted in a dominant negative manner to suppress NE upregulation by the WT G-CSF receptor. In luciferase reporter assays, the WT G-CSF receptor, but not the d715 mutant, activated a 1.8-kb fragment of the mouse Ela2 promoter. Significantly, forced expression of an SCN-associated NE mutant G185R caused premature apoptosis of differentiating 32D/WT cells in response to G-CSF with no significant effect on IL-3-stimulated survival. To address whether the d715 mutant may abolish the proapoptotic effect of the G185R mutant via suppressing its expression, we transfected 32D/WT and 32D/d715 cells with an expression construct in which the expression of the G185R mutant was driven by the 1.8-kb fragment of the Ela2 promoter. G-CSF treatment induced the expression of the G185R mutant and subsequent apoptosis in 32D/WT cells. In 32D/d715 cells, however, the expression of the G185R mutant was not induced by G-CSF and accordingly its proapoptotic activity was not evident. We propose that acquisition of the nonsense mutations in CSF3R may represent a mechanism utilized by the myeloid cells harboring the ELA2 mutations to evade the proapoptotic effect of the NE mutants. However, expression of the truncated G-CSF receptors has other biological consequences: they transduce strong proliferative signals but are defective in inducing granulocytic differentiation, which may initiate the leukemogenic process.

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Dispensable Role of Neutrophil Elastase in Survival and Differentiation of Human Myeloid Progenitor Cells

Blood ◽

10.1182/blood.v112.11.3561.3561 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3561-3561

Author(s):

Andrew A Aprikyan ◽

Vahagn Makaryan ◽

Qian Si ◽

Kelly Treonze ◽

Nara Markosyan ◽

...

Keyword(s):

Cell Survival ◽

Progenitor Cells ◽

Neutrophil Elastase ◽

Autosomal Dominant ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Wild Type ◽

Congenital Neutropenia ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

Abstract Severe congenital neutropenia (SCN) is a rare autosomal dominant or recessive disorder with a characteristic “maturation arrest” at the promyelocytic stage of differentiation in the bone marrow and extremely low level of neutrophils in peripheral circulation. SCN patients may evolve to develop myelodysplastic syndrome and acute myeloid leukemia (MDS/AML) with ~30% cumulative incidence of leukemia. Heterozygous mutations in the neutrophil elastase (NE, ELA2) gene have been identified in most of SCN patients with acquired and autosomal dominant inheritance (Dale et al., Blood 2000). It has been reported that accelerated apoptosis of bone marrow myeloid progenitor cells is the cellular mechanism of severe neutropenia in SCN. We and others also reported that expression of mutant elastase triggers impaired cell survival in human myeloid progenitor cells (Aprikyan et al., Exp Hem 2003; Massullo et al, Blood 2005; Kollner et al, Blood 2006; Grenda et al, Blood 2007). However, it remains unclear whether the abnormal cell survival and impaired myeloid differentiation in SCN is due to the gain-of-function or dominant negative effect of mutant NE. To answer these questions, we established tet-off HL-60 human myeloid progenitor cell lines with inducible expression of mutant or normal forms of NE and examined their survival and differentiation characteristics. Induced expression of del.145–152 mutant NE resulted in a significantly increased apoptosis of myeloid cells and a characteristic block of myeloid differentiation (p<0.05, n=4), thus closely recapitulating the human SCN phenotype. Importantly, induced overexpression of wild type NE in the myeloid progenitor cells did not alter the cell survival characteristics as determined by flow cytometry analyses of annexin V or DIOC6 stained cells (p>0.05, n=4). In addition, the overexpression of wild type NE had no effect on differentiation capacity of myeloid progenitor cells induced into granulocytic differentiation with retinoic acid compared with control cells expressing physiological levels of endogenous NE as determined by morphological evaluation of stained cells. Furthermore, nearly complete inhibition of NE proteolytic activity in control human myeloid progenitor cells expressing endogenous elastase with a cell-penetrant small molecule inhibitor of NE (Merck, NJ) also had no significant effect and impaired neither the cell survival nor their differentiation characteristics compared with control untreated cells (p>0.05, n=3). Thus, these data demonstrate that neutrophil elastase appears to be dispensable for the formation and differentiation of HL-60 human myeloid progenitor cells and accelerated apoptosis and impaired myeloid differentiation in SCN is attributable to a gain-of-function effect of pro-apoptotic mutant elastase. These data also suggest that small molecule inhibitors of NE may represent a promising therapy in severe congenital neutropenia.

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Abstract B126: A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo

10.1158/1535-7163.targ-19-b126 ◽

2019 ◽

Author(s):

Mark Albertella ◽

Wesley Blackaby ◽

Richard Fosbeary ◽

Alan Hendrick ◽

Dan Leggate ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Small Molecule ◽

Myeloid Leukemia ◽

Small Molecule Inhibitor ◽

Molecule Inhibitor ◽

Acute Myeloid

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A Model of Severe Congenital Neutropenia Reveals Signaling Pathways Mediating Mutant Elastase-Triggered Agranulocytosis.

Blood ◽

10.1182/blood.v106.11.3070.3070 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3070-3070

Author(s):

Andrew A. Aprikyan ◽

Tomas Vaisar ◽

Vahagn Makaryan ◽

Jay Heinecke

Keyword(s):

Bone Marrow ◽

Signaling Pathways ◽

Myelogenous Leukemia ◽

Severe Neutropenia ◽

Myeloid Differentiation ◽

Cellular Model ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Histone H2b ◽

Maturation Arrest

Abstract Severe congenital neutropenia (SCN; Kostmann’s syndrome or infantile genetic agranulocytosis) defines an inheritable hematopoietic disorder of impaired neutrophil production due to a “maturation arrest” at the promyelocytic stage of differentiation in the bone marrow. SCN patients have recurring severe infections and often develop acute myelogenous leukemia. We and others reported accelerated apoptosis and cell cycle arrest of bone marrow-derived myeloid progenitor cells in SCN patients with autosomal dominant and autosomal recessive inheritance. Heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease, are present in a majority of SCN patients, but not in healthy members of the family, thus indicating a key role of mutant NE in pathogenesis of this disorder. To date, there are no animal or cellular models of SCN as both the knock-in of mutant NE as well as the knock-out of normal NE failed to result in neutropenia phenotype in mice. The molecular mechanisms of mutant NE-mediated severe neutropenia remain largely unknown. We hypothesized that mutations in NE expose the protease to a new range of substrates. To explore this proposal, we established a cellular model of SCN based on tetracycline-regulated expression of mutant NE in human promyelocytic tet-off HL-60 cells that very closely recapitulated the human phenotype. Mutant NE expression resulted in a characteristic block of myeloid differentiation - the cellular hallmark of SCN. Expression of the mutant product was associated with a significant reduction in phosphatidylinosytol-3-kinase and phosphorylated PKB/Akt levels and an imbalance of anti-apoptotic Bcl-2 and pro-apoptotic Bax. These alterations contributed to observed dissipation of mitochondrial membrane potential as determined by FACS analysis, aberrant release of cytochrome C, and accelerated apoptosis. Marked changes in actin cytoskeleton that made the cells more rigid appeared to stem from a reduced level of alpha-actinin and elevated level of Rho GTPase. Immunoprecipitation of cell lysates with elastase-specific monoclonal antibodies followed by mass spectrometric analysis revealed that NE interacted with histone H2B, one of the key components of the nucleosome core of the chromatin. Interestingly, the expression level of histone H2B was substantially reduced in cells expressing mutant NE, therefore supporting the notion of altered substrate specificity of mutant NE. Thus, these observations provide the first evidence that mutant NE affects specific signaling pathways that lead to alterations in cytoskeleton and chromatin reorganization, subsequent apoptosis, and a block of myeloid differentiation in SCN. This cellular model of SCN should provide an invaluable tool for screening potential therapeutic agents capable of preventing maturation arrest and leukemogenesis in subjects suffering from severe congenital neutropenia.

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Abnormal Localization of Neutrophil Elastase in Patients with Severe Congenital Neutropenia.

Blood ◽

10.1182/blood.v108.11.1279.1279 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1279-1279

Author(s):

Takashi Sato ◽

Masakazu Habara ◽

Hiroki Kihara ◽

Hiroshi Kawaguchi ◽

Mizuka Miki ◽

...

Keyword(s):

Bone Marrow ◽

Dimensional Analysis ◽

Protein Trafficking ◽

Neutrophil Elastase ◽

Plasma Membranes ◽

Normal Subjects ◽

Precursor Cells ◽

Proteinase 3 ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia

Abstract Mutations in the ELA2 gene encoding neutrophil elastase (NE) in patients with severe congenital neutropenia (SCN) are involved in the pathogenesis of this disorder, possibly due to the abnormal protein trafficking and accelerated apoptosis of myeloid cells. In this study we precisely examined the localization of NE in neutrophils and myeloid precursor cells in bone marrow in patients with SCN using immunofluorescence microscopy equipped with three-dimensional analysis program. Three patients with SCN were enrolled in this study. All patients with SCN showed heterozygous mutation in the ELA2 gene. In normal subjects the pattern of localization of NE in mature neutrophils was almost similar to those of myeloperoxidase (MPO), proteinase 3, lysosomal associated membrane protein 2 (LAMP2). Administration of G-CSF to normal subjects did not affect the pattern of the localization of these proteins in neutrophils. In contrast, mature neutrophils elicited by the administration of G-CSF in patients with SCN NE predominantly localized to the plasma membranes. A small part of NE was detected in the cytoplasmic compartment. The pattern of localization of NE was significantly different from those of MPO, proteinase 3, and LAMP2 in SCN patients, suggesting the abnormal traffic of NE to granules. Adaptor proteins 3 (AP3) specifically shuttles transmembrane cargo proteins from the trans-Golgi to lysosomes. AP3 of myeloid progenitor cells enriched for CD33-positive cells in normal bone marrow was localized in both cytoplasm and plasma membranes. The localization pattern of AP3 was completely consistent with those of NE, MPO, and LAMP2. The localization of AP3 of promyelocytes in patients with SCN was observed in both plasma membranes and cytoplasm. This finding was completely similar to that in normal myeloid precursor cells. However, the localization of NE of promyelocytes in SCN patients was predominantly in plasma membrane. The figures merged apparently presented the different localization of NE and AP3. This result was confirmed by the 3-dimensional analysis with histogram. The localizations of other constituents of primary granules, MPO, poteinase 3, and LAMP2, were consistent with those of AP3. These observations suggest that the mislocalization of NE in myeloid precursor cells in SCN patients does not result from a generalized impairment of protein trafficking but is specific to the mutant NE. The abnormal localization of NE may be involved in the pathogenesis of SCN associated with ELA2 mutation.

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Axl blockade in vitro and in patients with high-risk MDS by the small molecule inhibitor BGB324.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.7059 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 7059-7059 ◽

Cited By ~ 1

Author(s):

Sonja Loges ◽

Isabel Ben Batalla ◽

Michael Heuser ◽

Nikolas Berenbrok ◽

Thomas Schroeder ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Adverse Events ◽

Small Molecule ◽

Small Molecule Inhibitor ◽

Loading Dose ◽

Bone Marrow Stroma ◽

Healthy Donors ◽

Molecule Inhibitor ◽

Marrow Stroma

7059 Background: The interplay with bone marrow stroma plays an important role in the pathobiology of MDS. Gas6 is secreted by mesenchymal bone marrow stroma cells and promotes survival and therapy resistance of AML cells expressing the Axl receptor. We hypothesized that inhibiting Axl by the small molecule inhibitor BGB324 might hold therapeutic potential in MDS. Methods: We investigated the inhibitory effect of BGB324 on primary bone marrow mononucleated cells (BMMNC) and mesenchymal stroma cells (MSC) from MDS patients in comparison to healthy donors. In the ongoing first-in-patient Phase 1a/b trial BGBC003 A standard 3 + 3 dose escalation study was performed to identify the maximum tolerated dose of BGB324 in patients with previously treated high risk MDS or AML. BGB324 was administered as an oral loading dose on days one and two followed by a reduced daily maintenance. Three dose levels were explored 400/100mg, 600/200mg and 900/300mg. Results: We found that BGB324 inhibited BMMNC from low- and high-risk MDS patients with an IC50 of 2.1 µM and 3.8 µM, respectively (n = 5). In comparison, BMNNC from healthy donors were resistant to BGB324 (IC50 9.4 µM, p < 0.05, n = 10). Axl expression was present in MSC isolated from the BM of MDS patients and BGB324 inhibited the proliferation of MSC from low- and high-risk MDS patients (IC50 2.5 µM and 2.7 µM, respectively; n = 7/5).To date, 3 patients with MDS were treated with 400 mg loading dose and 100 mg maintenance dose of BGB324. Therapy has been well-tolerated and the MTD has not yet been reached. The majority of adverse events reported have been Grade 1 and 2. The most common related adverse events are diarrhea and fatigue. One patient with MDS was treated for 80 weeks and experienced a PR. Evidence of target inhibition was demonstrated by almost complete inhibition of Axl phosphorylation accompanied by reduction in phosphoErk and phosphoAkt signalling at day 21 of treatment. Conclusions: BGB324 is well-tolerated and might represent a promising novel treatment approach in MDS. Safety and efficacy of BGB324 will be explored further in clinical trials. Clinical trial information: NCT02488408.

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