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•Dasatinib, asciminib, and prednisone can be safely combined for the dual targeting of BCR::ABL1 in Ph+acute leukemia.•Dasatinib and asciminib combination treatment has promising efficacy for the upfront treatment of Ph+ALL.

Clonal hematopoiesis (CH) results from somatic genomic alterations that drive clonal expansion of blood cells. Somatic gene mutations associated with hematologic malignancies detected in hematopoietic cells of healthy individuals, referred to as CH of indeterminate potential (CHIP), have been associated with myeloid malignancies, while mosaic chromosomal alterations (mCAs) have been associated with lymphoid malignancies. Here, we analyzed CHIP in 55,383 individuals and autosomal mCAs in 420,969 individuals with no history of hematologic malignancies in the UK Biobank and Mass General Brigham Biobank. We distinguished myeloid and lymphoid somatic gene mutations, as well as myeloid and lymphoid mCAs, and found both to be associated with risk of lineage-specific hematologic malignancies. Further, we performed an integrated analysis of somatic alterations with peripheral blood count parameters to stratify the risk of incident myeloid and lymphoid malignancies. These genetic alterations can be readily detected in clinical sequencing panels and used with blood count parameters to identify individuals at high risk of developing hematologic malignancies.

Malignant transformation of cells typically involves several genetic lesions, whose combined activity gives rise to cancer1. Here we analyse 1,148 patient-derived B-cell leukaemia (B-ALL) samples, and find that individual mutations do not promote leukaemogenesis unless they converge on one single oncogenic pathway that is characteristic of the differentiation stage of transformed B cells. Mutations that are not aligned with this central oncogenic driver activate divergent pathways and subvert transformation. Oncogenic lesions in B-ALL frequently mimic signalling through cytokine receptors at the pro-B-cell stage (via activation of the signal-transduction protein STAT5)2–4or pre-B-cell receptors in more mature cells (via activation of the protein kinase ERK)5–8. STAT5- and ERK-activating lesions are found frequently, but occur together in only around 3% of cases (P= 2.2 × 10−16). Single-cell mutation and phospho-protein analyses reveal the segregation of oncogenic STAT5 and ERK activation to competing clones. STAT5 and ERK engage opposing biochemical and transcriptional programs that are orchestrated by the transcription factors MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway comes at the expense of the principal oncogenic driver and reverses transformation. Conversely, deletion of divergent pathway components accelerates leukaemogenesis. Thus, persistence of divergent signalling pathways represents a powerful barrier to transformation, while convergence on one principal driver defines a central event in leukaemia initiation. Pharmacological reactivation of suppressed divergent circuits synergizes strongly with inhibition of the principal oncogenic driver. Hence, reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to enhance treatment responses.

•Prior to a bone marrow harvest, the provider reviews the step-by-step process, the risks and complications of the procedure and obtains consent for the procedure with the donor.•To perform an optimal bone marrow harvest, there are several considerations including: anesthesia, instrument choice, site selection, patient positioning, site preparation, operating room set-up, and the procedure itself.•Considerations for a safe discharge of a donor after a bone marrow harvest include pain control, absence of nausea and vomiting, adequate micturition, stable blood pressure, availability of a suitable caregiver and stable hemoglobin.•The bone marrow harvest process described in detail in this white paper ensures providers who perform bone marrow harvests provide the patient with the optimal stem cell product while achieving donor safety.•Throughout the bone marrow harvest procedure, donor safety is paramount.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton’s tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

Therapeutics that block kinases, transcriptional modifiers, immune checkpoints and other biological vulnerabilities are transforming cancer treatment. As a result, many patients achieve dramatic responses, including complete radiographical or pathological remission, yet retain minimal residual disease (MRD), which results in relapse. New functional approaches can characterize clonal heterogeneity and predict therapeutic sensitivity of MRD at a single-cell level. Preliminary evidence suggests that iterative detection, profiling and targeting of MRD would meaningfully improve outcomes and may even lead to cure.

Introduction:

Introduction:Oral ABL1 kinase inhibitors rapidly produce deep remissions in BCR::ABL1+ acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphoid blast crisis (CML-LBC). Second generation TKIs such as dasatinib (DAS) are more effective than imatinib (Foa et al. Blood2011) but patients may develop resistance. Asciminib (ASC), previously ABL001, is a STAMP (Specifically Targeting the ABL Myristoyl Pocket) allosteric ABL1 inhibitor that binds to a site spatially distinct from ATP-competitive TKIs. Combination treatment with an allosteric and an ATP-competitive TKI may deepen clinical responses and limit mutational resistance as supported by a cell line xenograft model of CML (Wylie et al. Nature2017) and patient-derived xenograft models of BCR::ABL1+ ALL. We hypothesized that dual ABL blockade with catalytic domain and allosteric inhibitors would be tolerable and active in BCR::ABL1+ ALL and CML-LBC.

Introduction:Minimal residual disease (MRD) detected by circulating tumor DNA (ctDNA) has emerged as a promising biomarker in diffuse large B-cell lymphoma (DLBCL). MAESTRO (minor-allele-enriched sequencing through recognition oligonucleotides) was recently developed to aid the detection of low-frequency mutations by enriching for mutant alleles using probes preferentially capturing single-nucleotide variants. We have extended this application - termed MAESTRO-Pool - to analyze personalized MRD variant detection within a cohort-level single assay. We demonstrate high sensitivity to detect MRD using MAESTRO-Pool and the detection of emergent mutations using targeted sequencing of the same samples.

IntroductionAdults with acute lymphoblastic leukemia (ALL), especially those who are older and/or have unfavorable disease biology frequently relapse. Inotuzumab ozogamicin (INO), an anti-CD22 monoclonal antibody covalently linked to calicheamicin, is approved for treatment of relapsed or refractory (R/R) CD22+ ALL. Although INO induces measurable residual disease negative (MRD-neg) complete remission (CR) in >75% of patients (pts), the duration of response (DOR) is limited (<6 months). Pre-clinical work demonstrates that malignant lymphoblasts are BCL2-dependent (Del Gaizo Moore Blood 2008). Initial results of the BCL2 inhibitor venetoclax (VEN) combination therapies for ALL are promising (Jain ASH 2019, Pullarkat Cancer Discov 2021). Due to distinct mechanisms of activity, non-overlapping toxicity, and possible synergy (Kirchhoff Blood 2021), we hypothesized that INO and VEN can be safely and effectively combined.

SUMMARY Until recently, the treatment of blood cancers has rested exclusively on chemotherapy, radiation and, in select cases, stem cell transplantation, giving rise to frequent and sometimes life-threatening side effects. The past 10 years have witnessed dramatic clinical advances due to the development of novel therapies, hereafter referred to as targeted therapies, which specifically inhibit molecules that are essential to the pathophysiology of individual malignancies. In this article, we will discuss the general concept of targeting signaling pathways in cancers and the limitations of this strategy, with a particular focus on the emergence of resistant cancer clones under the selective pressures exerted by targeted therapies. Finally, we will examine a number of targeted therapies with immediate application in contemporary clinical practice.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNA-like ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. However, the mechanisms by which these mutations disrupt granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the production of misfolded NE protein, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a specific ELA2 mutation correlated with its associated clinical phenotype. Consistent with the UPR model, expression of mutant NE in primary human granulocytic precursors increased expression of CHOP (DDITS) and induced apoptosis in a protease-independent fashion. Most strikingly, UPR activation and decreased NE protein expression were detected in primary granulocytic precursors from SCN patients. Collectively, these data provide strong support for a UPR model of SCN disease pathogenesis and place SCN in a growing list of human diseases caused by misfolded proteins.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. However, the mechanisms by which these mutations disrupt granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the production of misfolded NE protein, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a specific ELA2 mutation correlated with its associated clinical phenotype. Consistent with the UPR model, expression of mutant NE in primary human granulocytic precursors increased expression of CHOP (DDITS) and induced apoptosis in a protease-independent fashion. Most strikingly, UPR activation and decreased NE protein expression were detected in primary granulocytic precursors from SCN patients. Collectively, these data provide strong support for a UPR model of SCN disease pathogenesis and place SCN in a growing list of human diseases caused by misfolded proteins.

IntroductionOral tyrosine kinase inhibitors (TKIs) that inhibit the constitutively active ABL1 kinase have improved outcomes for BCR-ABL1+ acute lymphoblastic leukemia (ALL) and decreased reliance on cytotoxic chemotherapy (ctx). Later generation, more potent TKIs such as dasatinib (DAS) are particularly effective (Foa et al. Blood 2011; Foa et al. N Eng J Med 2020). ABL001 (asciminib) is a STAMP (Specifically Targeting the ABL Myristoyl Pocket) allosteric inhibitor of ABL1 that binds to a site spatially distinct from all approved ATP-competitive TKIs. Combination treatment with an allosteric and an ATP-competitive TKI may deepen clinical responses and limit mutational resistance as supported by a cell line xenograft model of CML (Wylie et al. Nature 2017) and patient-derived xenograft models of BCR-ABL1+ ALL. We hypothesized that dual ABL blockade with catalytic domain and allosteric inhibitors would be tolerable and effective in BCR-ABL1+ ALL.

Background:The concept of multi-step carcinogenesis (Fearon and Vogelstein 1990) suggests that acquisition of mutations in addition to an existing set of mutations invariably accelerates tumor-progression. In colorectal cancer and many other cancer types, activation of multiple distinct oncogenic pathways is required for the development of invasive cancer. Here, we examined this paradigm for genetic lesions in B-ALL and 13 other cancer types.

Wiita: UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Izraeli:sightdx: Consultancy; novartis: Honoraria; prime oncology: Speakers Bureau. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding.

IntroductionThe BCR-ABL1 gene fusion is the most common molecular aberration in adult ALL and confers an adverse prognosis. The incorporation of ABL1 tyrosine kinase inhibitors (TKIs) into ALL chemotherapy regimens has dramatically improved outcomes for BCR-ABL1+ ALL. Subsequently, reduced-intensity TKI-based regimens have been shown to be as effective and less toxic than intensive TKI-based regimens (Foa et al. Blood 2011; Chalandon et al. Blood 2015). Asciminib (ABL001) is a potent, specific allosteric inhibitor of ABL1 that binds to a site spatially distinct from the catalytic domain. Asciminib was developed for efficacy against BCR-ABL1 mutations conferring resistance to standard TKIs. Preclinical studies demonstrated that the combination of asciminib and TKI leads to sustained disease control in a cell line xenograft model of BCR-ABL1+ CML (Wylie et al. Nature 2017). We also observed that the addition of asciminib to TKI significantly prolongs survival in patient-derived xenograft models of BCR-AB1L+ ALL, even in the context of a pre-existing subclonal T315I mutation (unpublished). An ongoing phase I Novartis study (NCT02081378) has shown safety and efficacy of asciminib alone and in combination with TKIs in relapsed/refractory (R/R) BCR-ABL1+ ALL and CML. We hypothesized that dual ABL blockade with catalytic domain and allosteric inhibitors would be tolerable and effective in older adults with BCR-ABL1+ ALL.

Introduction: Assays are needed to define the susceptibility of heterogeneous primary human tumor cells. We previously showed that single-cell mass accumulation rate (MAR) - conceptually similar to growth rate - can accurately define drug sensitivity ex vivo(Stevens et al. Nature Biotechnology,2016). This live-cell assay is performed using a suspended microchannel resonator (SMR), which maintains cell viability and permits the downstream application of single-cell whole transcriptome sequencing (scRNA-seq) to identify molecular correlates of drug response. By comparing leukemias exposed to antineoplastic therapy or vehicle in vivo, we sought to identify unique transcriptional signatures that, when coupled with single-cell biophysical properties, provide a more nuanced view of therapeutic susceptibility.

Jacobson: Kite: Membership on an entity's Board of Directors or advisory committees. Armand:Pfizer: Research Funding; Sequenta Inc: Research Funding; Merck: Consultancy, Research Funding; Roche: Research Funding; Infinity Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Shipp:Bristol-Myers Squibb: Consultancy, Research Funding; Cell Signaling: Honoraria; Merck, Gilead, Takeda: Other: Scientific Advisory Board; Bayer: Research Funding. Fisher:Pharmacyclics: Consultancy. Weinstock:Novartis: Consultancy, Research Funding.

Barzaghi-Rinaudo: Novartis Institutes for Biomedical Research: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding. Jeay:Novartis: Employment, Equity Ownership. Wuerthner:Novartis: Employment, Equity Ownership. Halilovic:Novartis: Employment, Equity Ownership.

Davids: Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees.

Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Etchin:Karyopharm: Research Funding. Lane:Stemline Therapeutics, Inc.: Research Funding. Stone:Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Merck: Consultancy; Karyopharm: Consultancy; Roche/Genetech: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy.

No relevant conflicts of interest to declare.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Most cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. Herein, we report the preliminary description of a novel transgenic mouse line that expresses G192X Ela2, reproducing the G193X ELA2 mutation found in some patients with SCN. The G192X mutation was introduced into the murine Ela2 locus by homologous recombination in embryonic stem cells. Heterozygous or homozygous G192 Ela2 “knock-in” mice were healthy with no apparent developmental defect. While expression of Ela2 mRNA was normal, no mature NE protein was detected in the neutrophils of homozygous G192X Ela2 mice. However, in granulocytic precursors (mainly promyelocytes/myelocytes) a small amount of heavily glycosylated mutant NE protein was detected. Together, these observations suggest that G192X NE protein is retained in the endoplasmic reticulum (ER) and rapidly degraded. Consistent with ER stress and induction of the UPR, a significant increase in BiP/GRP78 and ATF6 mRNA expression in mutant granulocytic precursors were observed. Surprisingly, G192X Ela2 mice have normal basal granulopoiesis. The number of circulating neutrophils, granulocytic differentiation in the bone marrow, and number and cytokine responsiveness of myeloid progenitors were comparable to wild type mice. In summary, the G192X Ela2 mice appear to reproduce the NE protein misfolding and UPR activation observed in human SCN granulocytic precursors. However, expression of G192X Ela2 is not sufficient to disrupt basal granulopoiesis in mice. Studies of stress granulopoiesis are underway.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of ELA2, encoding neutrophil elastase (NE), are present in approximately 50% of cases of SCN. To date, 52 distinct, mostly missense, mutations of ELA2 have been identified. The diversity of ELA2 mutations in SCN and lack of consistent effect of these mutations on NE enzyme activity led us to hypothesize that structural rather than functional perturbations in the NE protein might be responsible for the disruption in granulopoiesis. Specifically, we hypothesized that a shared feature of the different NE mutants is their propensity to misfold. In this model, accumulation of misfolded NE in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) and ultimately leads to apoptosis of granulocytic precursors. The UPR is an adaptive cellular program that ameliorates protein-folding defects in the ER by attenuating most new translation and increasing transcription of genes involved in productive refolding and ER-associated degradation of misfolded proteins. However, if the ER stress is severe and ER homeostasis cannot be restored, the UPR triggers cell apoptosis. In support of this model, we and others previously showed that expression of mutant but not wild-type NE in myeloid cells induces UPR activation. Moreover, expression of mutant NE induced apoptosis in a protease-independent fashion. Finally, we showed that BiP/GRP78 mRNA expression and XBP-1 splicing, two classic markers of UPR activation, were induced in primary SCN granulocytic precursors. These data, while supporting the UPR model of disease pathogenesis, are limited in their scope. Previous studies have shown that activation of the UPR induces the transcription of a characteristic set of genes involved in ER homeostasis. In the present study, we performed RNA profiling on primary SCN granulocytic precursors to determine whether these cells displayed a transcriptional profile consistent with the UPR. Fresh bone marrow samples were obtained from 8 patients with SCN (7 with ELA2 mutations), 5 healthy controls, 2 healthy controls treated with G-CSF for 5 days, or 4 patients with Shwachman-Diamond syndrome (as a control for stress response genes induced by neutropenia, per se). In addition, bone marrow from two healthy controls were cultured for 24 hours with or without tunicamycin (an established activator of the UPR). In each case, CD15+CD16lowCD14-CD9- cells were isolated by sorting to obtain a cell population highly enriched for promyelocytes/myelocytes (>85% in all cases). RNA profiling was performed using the Affymetrix HG U133 Plus 2.0 Array. Remarkably, the primary SCN granulocytic precursors, but not the other cohorts, exhibit a transcriptional program similar to that of normal granulocytic precursors treated with tunicamycin. Specifically, we detected similar upregulation of the following classes of genes that are transcriptionally activated by the UPR: ER resident chaperones (subclasses HSP40, HSP70, and HSP90), foldases (disulfide isomerase and peptidylprolyl isomerase subclasses), and mediators of apoptosis (CHOP/DDIT3 and pro-apoptotic members of the Bcl-2 family). These data provide strong new evidence in support of the UPR model of SCN disease pathogenesis.

In order to better understand the pathogenesis of acute promyelocytic leukemia (APL, FAB M3), we sought to determine its gene expression signature by comparing the expression profiles of 14 APL samples to that of other AML subtypes (M0, M1, M2, M4, n=62) and to fractionated normal whole bone marrow cells (CD34 cells, promyelocytes, PMNs, n=5 each). We used ANOVA and SAM (Significance Analysis of Microarrays) to select genes that were highly expressed in APL cells and that displayed low to no expression in other AML subtypes. The APL signature was then further refined by filtering genes whose expression in APL was not significantly different from that of normal promyelocytes, yielding 1121 annotated genes that reliably distinguish APL from the other FAB subtypes using unsupervised hierarchical clustering, both in training and validation datasets. Fold change differences in expression between M3 and other AML FAB classes were striking, for example: GABRE 35.4, HGF 21.3, ANXA8 21.3, PTPRG 16.9, PTGDS 12.1, PPARG 11.1, STAB1 9.8. A large proportion of the APL versus other FAB dysregulome was recapitulated when we compared APL expression to that of the normal pattern of myeloid development. We identified 733 annotated genes with significantly different expression in APL versus normal myeloid cell fractions. These dysregulated genes were assigned to 4 classes: persistently expressed CD34 cell-specific genes, repressed promyelocyte-specific genes, prematurely expressed neutrophil-specific genes and genes with high expression in APL and low/no expression in normal myeloid cell fractions. Expression differences in several of the most dysregulated genes were validated by qRT-PCR. We then examined the expression of the APL signature genes in myeloid cell lines and tumors from a murine APL model. The bona fide M3 signature was not apparent in resting NB4 cells (which contain t(15;17), and which express PML-RARA), nor in PR-9 cells following Zn induction of PML-RARA expression, suggesting that neither cell line accurately models the gene expression signature of primary APL cells. Most of the nodal genes of the mCG-PML-RARA murine APL dysregulome (Yuan, et al, 2007) are similarly dysregulated in human M3 cells; however, the human and mouse dysregulomes do not completely coincide. Finally, we have begun investigating which APL signature genes are direct transcriptional targets of PML-RARA. The promoters of the APL signature genes were analyzed for the presence of known PML-RARA binding sites using multiple computational methods. The analyses demonstrated that several transcription factors (EBF3, TWIST1, SIX3, PPARG) have putative retinoic acid response elements (RAREs) in their upstream regulatory regions. Additionally, we examined the promoters of some of the most upregulated genes (HGF, PTGDS, STAB1) for known consensus sites of these transcription factors, and found that all have putative binding sites for at least one. These results suggest that PML-RARA may initiate a transcriptional cascade that relies not only on its own activity, but also on the actions of downstream transcription factors. In summary, our studies indicate that primary APL cells have a gene expression signature that is consistent and highly reproducible, but different from commonly used human APL cell lines and a mouse model of APL. The molecular mechanisms that govern this unique signature are currently under investigation.

Severe congenital neutropenia (SCN) is characterized by severe chronic neutropenia and promyelocyte accumulation in the bone marrow. Mutations in the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and nearly all cases of cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. To date, 47 distinct, mostly missense, mutations have been reported in patients with CN or SCN; most segregate with one phenotype, suggesting a genotype-phenotype correlation. While genetic studies suggest that ELA2 mutations act in a dominant, cell-intrinsic fashion to disrupt granulopoiesis, the molecular mechanisms by which they do so are unknown. Given the functional heterogeneity of NE mutants in SCN, we hypothesized that ELA2 mutations disrupt granulopoiesis by leading to the production of NE proteins that misfold, activate the unfolded protein response (UPR), and ultimately trigger apoptosis in granulocytic precursors. The UPR is a well-characterized cellular program that acts to ameliorate the accumulation of misfolded proteins in the endoplasmic reticulum (ER) via general attenuation of translation initiation, upregulated expression of ER resident protein chaperones, and increased ER-associated degradation (ERAD) of misfolded proteins. Persistence of the folding defect promotes apoptosis. We previously showed that expression of mutant NE induces BiP/GRP78 gene expression, a classic biochemical marker of the UPR, and impairs clonogenic capacity in a myelomonocytic cell line. To further explore the UPR hypothesis, we employed a transient transfection assay in which granulocytic precursors cultured from human cord blood-derived CD34+ cells express wild type or mutant forms of NE associated with SCN (V72M, G185R, G192pter), CN (R191Q), or both SCN and CN (P110L). Here we show that: Expression of SCN-related NE mutants but not R191Q NE induces BiP mRNA expression and XBP1 mRNA splicing, classic markers of the UPR; The degree of the UPR induced by each mutant in our study roughly correlates with the severity of its associated clinical phenotype. Notably, G185R NE, associated with the most severe clinical phenotype, induces the greatest BiP expression; Expression of SCN-related NE mutants is associated with increased apoptosis; and Protease-deficient double mutant forms of NE still induce the UPR and trigger apoptosis, suggesting that mutant NE disrupts granulopoiesis by a protease-independent mechanism. We next analyzed primary granulocytic precursors from 6 ELA2-positive SCN patients and 5 healthy donors and detected a 5.7-fold increase in BiP mRNA expression (p=.06) and a 2.5-fold increase in XBP1 mRNA splicing (p=.03) in the SCN samples. In addition, confocal microscopy of normal and ELA2-positive SCN bone marrow cells stained for NE reveals a marked reduction in NE expression in SCN cells, consistent with the UPR hypothesis. Together, these data strongly support a UPR model of SCN disease pathogenesis, placing SCN in a growing list of human diseases caused by misfolded proteins. More importantly, SCN represents the first known case of a congenital disorder caused by UPR-induced apoptosis.

Severe congenital neutropenia (SCN) is characterized by severe chronic neutropenia and promyelocyte accumulation in the bone marrow. Mutations in the ELA2gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and nearly all cases of cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. To date, 47 distinct, mostly missense, mutations have been reported in patients with CN or SCN; most segregate with one phenotype, suggesting a genotype-phenotype correlation. While genetic studies suggest that ELA2mutations act in a dominant, cell-intrinsic fashion to disrupt granulopoiesis, the molecular mechanisms by which they do so are unknown. Given the functional heterogeneity of NE mutants in SCN, we hypothesized that ELA2mutations disrupt granulopoiesis by leading to the production of NE proteins that misfold, activate the unfolded protein response (UPR), and ultimately trigger apoptosis in granulocytic precursors. The UPR is a well-characterized cellular program that acts to ameliorate the accumulation of misfolded proteins in the endoplasmic reticulum (ER) via general attenuation of translation initiation, upregulated expression of ER resident protein chaperones, and increased ER-associated degradation (ERAD) of misfolded proteins. Persistence of the folding defect promotes apoptosis.