Delayed normalization of ADAMTS13 activity in acute thrombotic thrombocytopenic purpura in the caplacizumab era.

The benefits of caplacizumab in acute immune-mediated thrombotic thrombocytopenic purpura (iTTP) are well established. We identified a delayed normalization of a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) activity (>30%) in a subgroup treated with caplacizumab, not evident in the precaplacizumab era. Patients treated with caplacizumab (n = 64) achieved ADAMTS13 activity >30% at median 31 days after plasma exchange (PEX), compared with 11.5 days in the noncaplacizumab group (n = 50, P = .0004). Eighteen of 64 (28%) patients treated with caplacizumab had ADAMTS13 activity <10% at stopping caplacizumab with a longer time to ADAMTS13 activity >30% (median, 139 days after completing PEX). Eighteen of 64 (28%) patients receiving extended caplacizumab (31-58 days) failed to achieve ADAMTS13 activity >30% at the time of caplacizumab cessation, compared with 4 of 47 (8.5%) historical controls at a similar timepoint (30 + 28 days, P < .0001). Failure to achieve ADAMTS13 activity >30% within 30 + 28 days was 6 times more likely with caplacizumab (odds ratio, 6.3; P = .0006). ADAMTS13 antigen <30% at caplacizumab cessation was associated with increased iTTP recurrence (4/10 vs 0/9 in patients with ADAMTS13 antigen ≥30%). Admission anti-ADAMTS13 immunoglobulin G (IgG) antibody level did not predict recurrence. Anti-ADAMTS13 IgG antibody levels, immunosuppression, and ethnicity did not account for differences in ADAMTS13 activity response. ADAMTS13 antigen levels ≥30% may be useful to guide stopping caplacizumab therapy after extended use with ADAMTS13 activity <10%. The reason for delayed ADAMTS13 normalization is unclear and requires further investigation.

Long-term risk of relapse in immune-mediated thrombotic thrombocytopenic purpura and the role of anti-CD20 therapy.

Disease relapse is recognized as a risk in immune-mediated thrombotic thrombocytopenic purpura (iTTP) after treatment of the acute presenting episode. Identification of patients at risk of relapse and its patterns are yet to be clearly established. We reviewed patients with iTTP having had >3 years of follow-up over 10 years in the United Kingdom to identify patient characteristics for relapse, assess relapse rates and patterns, and response to anti-CD20 therapy in those with a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) relapses (ADAMTS13 activity of <20% without thrombocytopenia). We identified 443 patients demonstrating relapse rates of 40% at 5-year follow-up. At 10-year follow-up, no difference in relapse was observed irrespective of whether rituximab was used at acute presentation (P = .39). Black Caribbean ethnicity increased the risk of disease relapse in the British population. There was a distinct population of patients (6%) that relapsed early with subsequent frequent relapses occurring on average within 2 years (average time to relapse in subgroup, 1.7 years). Overall, nearly 60% of relapses described were ADAMTS13 relapses, with subsequent treatment reducing the risk of progression to clinical relapses. We demonstrate that iTTP diagnosed in the latter part of the study period had lower rates of clinical relapses (22.6% vs 11.1%, P = .0004) with the advent of regular monitoring and preemptive rituximab. In ADAMTS13 relapses, 96% responded to anti-CD20 therapy, achieving ADAMTS13 activity of >20%. Anti-CD20 therapy was demonstrated to be an effective long-term treatment regardless of relapse pattern and there was no loss of this treatment response after subsequent treatment episodes.

HMGA1 chromatin regulators induce transcriptional networks involved in GATA2 and proliferation during MPN progression.

Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.

Real-world experience with caplacizumab in the management of acute TTP.

The cornerstone of life-saving therapy in immune-mediated thrombotic thrombocytopenic purpura (iTTP) has been plasma exchange (PEX) combined with immunomodulatory strategies. Caplacizumab, a novel anti-von Willebrand factor nanobody trialed in 2 multicenter randomized controlled trials (RCTs) leading to European Union and US Food and Drug Administration approval, has been available in the United Kingdom (UK) through a patient access scheme. Data were collected retrospectively from 2018 to 2020 for 85 patients (4 children) receiving caplacizumab from 22 UK hospitals. Patient characteristics and outcomes in the real-world clinical setting were compared with caplacizumab trial end points and historical outcomes in the precaplacizumab era. Eighty-four of 85 patients received steroid and rituximab alongside PEX; 26% required intubation. Median time to platelet count normalization (3 days), duration of PEX (7 days), and hospital stay (12 days) were comparable with RCT data. Median duration of PEX and time from PEX initiation to platelet count normalization were favorable compared with historical outcomes (P < .05). Thrombotic thrombocytopenic purpura (TTP) recurred in 5 of 85 patients; all had persistent ADAMTS13 activity < 5 IU/dL. Of 31 adverse events in 26 patients, 17 of 31 (55%) were bleeding episodes, and 5 of 31 (16%) were thrombotic events (2 unrelated to caplacizumab); mortality was 6% (5/85), with no deaths attributed to caplacizumab. In 4 of 5 deaths, caplacizumab was introduced >48 hours after PEX initiation (3-21 days). This real-world evidence represents the first and largest series of TTP patients, including pediatric patients, receiving caplacizumab outside of clinical trials. Representative of true clinical practice, the findings provide valuable information for clinicians treating TTP globally.

The use of obinutuzumab and ofatumumab in the treatment of immune thrombotic thrombocytopenic purpura.

Rituximab, an anti-CD20 monoclonal antibody, can be used to treat immune thrombotic thrombocytopenic purpura (iTTP) during acute presentation or disease relapse. Undesirable side-effects include severe hypersensitivity reactions, particularly anaphylaxis and rituximab-induced serum sickness, with a minority not maintaining a response to treatment. Alternative humanised anti-CD20 treatments, obinutuzumab and ofatumumab, have been used. A review of the UK TTP Registry showed 15 patients received these drugs over 26 treatment episodes (eight obinutuzumab and 18 ofatumumab). Indications for alternative anti-CD20 treatment were severe infusion-related reactions, acute rituximab-induced serum sickness and a short duration of disease remission. All patients achieved disease remission (ADAMTS13 [A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13] activity ≥30 iu/dl) after a median 15 days and 92% of episodes achieved complete remission (≥60 iu/dl). Seven patients required further treatment for disease relapse with a median relapse-free survival of 17.4 months. All patients continued to respond to re-treatment with the preceding drug when relapse occurred. There were four adverse events in 26 treatment episodes (15%) - two infections and two infusion reactions. These results suggest that obinutuzumab and ofatumumab may be considered as an alternative option to rituximab in the treatment of iTTP with a comparable safety profile, absence of significant hypersensitivity reactions and sustained normalisation of ADAMTS13.

Identification of a novel genetic locus associated with immune-mediated thrombotic thrombocytopenic purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare, life-threatening disorder, mediated through severe ADAMTS13 deficiency causing multi-system micro-thrombi formation, and has specific human leukocyte antigen associations. We undertook a large genome-wide association study to investigate additional genetically distinct associations in iTTP. We compared two iTTP patient cohorts with controls, following standardized genome-wide quality control procedures for single-nucleotide polymorphisms and imputed HLA types. Associations were functionally investigated using expression quantitative trait loci (eQTL), and motif binding prediction software. Independent associations consistent with previous findings in iTTP were detected at the HLA locus and in addition a novel association was detected on chromosome 3 (rs9884090, P=5.22x10-10, odds ratio 0.40) in the UK discovery cohort. Meta-analysis, including the French replication cohort, strengthened the associations. The haploblock containing rs9884090 is associated with reduced protein O-glycosyltransferase 1 (POGLUT1) expression (eQTL P<0.05), and functional annotation suggested a potential causative variant (rs71767581). This work implicates POGLUT1 in iTTP pathophysiology and suggests altered post-translational modification of its targets may influence disease susceptibility.

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Parsaclisib Is a Next-Generation Phosphoinositide 3-Kinase δ Inhibitor with Reduced Hepatotoxicity and Potent Antitumor and Immunomodulatory Activities in Models of B-Cell Malignancy.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)δ inhibitors in B-cell malignancies is hampered by hepatotoxicity, requiring dose reduction, treatment interruption, and/or discontinuation of therapy. In addition, potential molecular mechanisms by which resistance to this class of drugs occurs have not been investigated. Parsaclisib (INCB050465) is a potent and selective next-generation PI3Kδ inhibitor that differs in structure from first-generation PI3Kδ inhibitors and has shown encouraging anti-B-cell tumor activity and reduced hepatotoxicity in phase 1/2 clinical studies. Here, we present preclinical data demonstrating parsaclisib as a potent inhibitor of PI3Kδ with over 1000-fold selectivity against other class 1 PI3K isozymes. Parsaclisib directly blocks PI3K signaling-mediated cell proliferation in B-cell lines in vitro and in vivo and indirectly controls tumor growth by lessening immunosuppression through regulatory T-cell inhibition in a syngeneic lymphoma model. Diffuse large B-cell lymphoma cell lines overexpressing MYC were insensitive to proliferation blockade via PI3Kδ signaling inhibition by parsaclisib, but their proliferative activities were reduced by suppression of MYC gene transcription. Molecular structure analysis of the first- and next-generation PI3Kδ inhibitors combined with clinical observation suggests that hepatotoxicity seen with the first-generation inhibitors could result from a structure-related off-target effect. Parsaclisib is currently being evaluated in multiple phase 2 clinical trials as a therapy against various hematologic malignancies of B-cell origin (NCT03126019, NCT02998476, NCT03235544, NCT03144674, and NCT02018861). SIGNIFICANCE STATEMENT: The preclinical properties described here provide the mechanism of action and support clinical investigations of parsaclisib as a therapy for B-cell malignancies. MYC overexpression was identified as a resistance mechanism to parsaclisib in DLBCL cells, which may be useful in guiding further translational studies for the selection of patients with DLBCL who might benefit from PI3Kδ inhibitor treatment in future trials. Hepatotoxicity associated with first-generation PI3Kδ inhibitors may be an off-target effect of that class of compounds.

Genetic Identification of Two Novel Loci Associated with Steroid-Sensitive Nephrotic Syndrome.

BACKGROUND: Steroid-sensitive nephrotic syndrome (SSNS), the most common form of nephrotic syndrome in childhood, is considered an autoimmune disease with an established classic HLA association. However, the precise etiology of the disease is unclear. In other autoimmune diseases, the identification of loci outside the classic HLA region by genome-wide association studies (GWAS) has provided critical insights into disease pathogenesis. Previously conducted GWAS of SSNS have not identified non-HLA loci achieving genome-wide significance. METHODS: In an attempt to identify additional loci associated with SSNS, we conducted a GWAS of a large cohort of European ancestry comprising 422 ethnically homogeneous pediatric patients and 5642 ethnically matched controls. RESULTS: The GWAS found three loci that achieved genome-wide significance, which explain approximately 14% of the genetic risk for SSNS. It confirmed the previously reported association with the HLA-DR/DQ region (lead single-nucleotide polymorphism [SNP] rs9273542, P=1.59×10-43; odds ratio [OR], 3.39; 95% confidence interval [95% CI], 2.86 to 4.03) and identified two additional loci outside the HLA region on chromosomes 4q13.3 and 6q22.1. The latter contains the calcium homeostasis modulator family member 6 gene CALHM6 (previously called FAM26F). CALHM6 is implicated in immune response modulation; the lead SNP (rs2637678, P=1.27×10-17; OR, 0.51; 95% CI, 0.44 to 0.60) exhibits strong expression quantitative trait loci effects, the risk allele being associated with lower lymphocytic expression of CALHM6. CONCLUSIONS: Because CALHM6 is implicated in regulating the immune response to infection, this may provide an explanation for the typical triggering of SSNS onset by infections. Our results suggest that a genetically conferred risk of immune dysregulation may be a key component in the pathogenesis of SSNS.

Comparison of Rituximab originator (MabThera) to biosimilar (Truxima) in patients with immune-mediated thrombotic thrombocytopenic purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is an acute, multisystem thrombotic microangiopathy mediated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) autoantibodies. Immunosuppression with anti-CD20 therapy is the mainstay of treatment. MabThera's patent has now expired and biosimilars have been approved. Eighty-four consecutive patient episodes over 2 years, prior to and following our switch to Truxima are presented. Day 1 (D1), Day 28 (D28) and 3-month platelet counts, ADAMTS13 activity, and CD19 levels, adverse reactions and infective complications were recorded. Platelet counts were not significantly different between acute MabThera and Truxima treatment (D1 P = 0.085, D28 P = 0.77, 3 months P = 0.71) and electively (D1 P = 0.79, D28 P = 0.68, 3 months P = 0.99). ADAMTS13 recovery also was not significantly different acutely (D1 P = 0.99, D28 P = 0.27, 3 months P = 0.26) and electively (D1 P = 0.59, D28 P = 0.61, 3 months P = 0.34). CD19% depletion at D1 and 3 months was not significantly different acutely (D1 P = 0.52, 3 months P = 0.56) and electively (D1 P = 0.22, 3 months P = 0.19). Infusion reactions and infective complications were comparable with both therapies. This is the first series of the Rituximab biosimilar Truxima to be reported in iTTP, demonstrating equivalence to MabThera in terms of ADAMTS13 recovery, CD19 depletion, and platelet count at D28 and 3 months post-administration, with comparable infusion and infective complications. The financial benefit of the biosimilar anti-CD20 is considerable.

The BET inhibitor INCB054329 reduces homologous recombination efficiency and augments PARP inhibitor activity in ovarian cancer.

OBJECTIVE: Homologous recombination (HR)-proficient ovarian tumors have poorer clinical outcomes and show resistance to poly ADP ribose polymerase inhibitors (PARPi). A subset of HR-proficient ovarian tumors show amplification in bromodomain and extra-terminal (BET) genes such as BRD4. We aimed to test the hypothesis that BRD4 inhibition sensitizes ovarian cancer cells to PARPi by reducing HR efficiency and increasing DNA damage. METHODS: HR-proficient ovarian cancer cell lines (OVCAR-3, OVCAR-4, SKOV-3, UWB1.289+BRCA1) were treated with BRD4-targeting siRNA, novel (INB054329, INCB057643) and established (JQ1) BET inhibitors (BETi) and PARPi (olaparib, rucaparib). Cell growth and viability were assessed by sulforhodamine B assays in vitro, and in SKOV-3 and ovarian cancer patient-derived xenografts in vivo. DNA damage and repair (pH2AX, RAD51 and BRCA1 foci formation, and DRGFP HR reporter activity), apoptosis markers (cleaved PARP, cleaved caspase-3, Bax) and proliferation markers (PCNA, Ki67) were assessed by immunofluorescence and western blot. RESULTS: In cultured cells, inhibition of BRD4 by siRNA or INCB054329 reduced expression and function of BRCA1 and RAD51, reduced HR reporter activity, and sensitized the cells to olaparib-induced growth inhibition, DNA damage induction and apoptosis. Synergy was observed between all BETi tested and PARPi. INCB054329 and olaparib also co-operatively inhibited xenograft tumor growth, accompanied by reduced BRCA1 expression and proliferation, and increased apoptosis and DNA damage. CONCLUSIONS: These results provide strong rationale for using BETi to extend therapeutic efficacy of PARPi to HR-proficient ovarian tumors and could benefit a substantial number of women diagnosed with this devastating disease.

Lactate Utilization Enables Metabolic Escape to Confer Resistance to BET Inhibition in Acute Myeloid Leukemia.

Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment. SIGNIFICANCE: Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML. See related commentary by Boët and Sarry, p. 950.

Organisation and delivery of a dedicated multidisciplinary prone ventilation team in the intensive care unit: Strategies and lessons from COVID-19.

BACKGROUND: COVID-19 placed immense strain on healthcare systems, necessitating innovative responses to the surge of critically ill patients, particularly those requiring mechanical ventilation. In this report, we detail the establishment of a dedicated critical care prone positioning team at University Hospital Southampton in response to escalating demand for prone positioning during the initial wave of the pandemic. METHODS: The formation of a prone positioning team involved meticulous planning and collaboration across disciplines to ensure safe and efficient manoeuvrers. A comprehensive training strategy, aligned with national guidelines, was implemented for approximately 550 staff members from a diverse background. We surveyed team members to gain insight to the lived experience. RESULTS: A total of 78 full-time team members were recruited and successfully executed over 1200 manoeuvres over an eight-week period. Our survey suggests the majority felt valued and expressed pride and willingness to participate again should the need arise. CONCLUSION: The rapid establishment and deployment of a dedicated prone positioning team may have contributed to both patient care and staff well-being. We provide insight and lessons that may be of value for future respiratory pandemics. Future work should explore objective clinical outcomes and long-term sustainability of such services.

Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.

HOXA9 is required for survival in human MLL-rearranged acute leukemias.

Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes, including HOXA9. Here, we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias using RNA interference. Gene expression profiling after HOXA9 suppression demonstrated co-down-regulation of a program highly expressed in human MLL-AML and murine MLL-leukemia stem cells, including HOXA10, MEIS1, PBX3, and MEF2C. We demonstrate that HOXA9 depletion in 17 human AML/ALL cell lines (7 MLL-rearranged, 10 MLL-germline) induces proliferation arrest and apoptosis specifically in MLL-rearranged cells (P = .007). Similarly, assessment of primary AMLs demonstrated that HOXA9 suppression induces apoptosis to a greater extent in MLL-rearranged samples (P = .01). Moreover, mice transplanted with HOXA9-depleted t(4;11) SEMK2 cells revealed a significantly lower leukemia burden, thus identifying a role for HOXA9 in leukemia survival in vivo. Our data indicate an important role for HOXA9 in human MLL-rearranged leukemias and suggest that targeting HOXA9 or downstream programs may be a novel therapeutic option.

A Humanized Animal Model Predicts Clonal Evolution and Therapeutic Vulnerabilities in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology. SIGNIFICANCE: Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation. This article is highlighted in the In This Issue feature, p. 2945.