Hematology
Abstract
Acute myeloid leukemia (AML) patients with NPM1 mutations demonstrate a superior response to standard chemotherapy treatment. Our previous work has shown that these favorable outcomes are linked to the cytoplasmic relocalization and inactivation of FOXM1 driven by mutated NPM1. Here, we went on to confirm the important role of FOXM1 in increased chemoresistance in AML. A multiinstitution retrospective study was conducted to link FOXM1 expression to clinical outcomes in AML. We establish nuclear FOXM1 as an independent clinical predictor of chemotherapeutic resistance in intermediate-risk AML in a multivariate analysis incorporating standard clinicopathologic risk factors. Using colony assays, we show a dramatic decrease in colony size and numbers in AML cell lines with knockdown of FOXM1, suggesting an important role for FOXM1 in the clonogenic activity of AML cells. In order to further prove a potential role for FOXM1 in AML chemoresistance, we induced an FLT3-ITD–driven myeloid neoplasm in a FOXM1-overexpressing transgenic mouse model and demonstrated significantly higher residual disease after standard chemotherapy. This suggests that constitutive overexpression of FOXM1 in this model induces chemoresistance. Finally, we performed proof-of-principle experiments using a currently approved proteasome inhibitor, ixazomib, to target FOXM1 and demonstrated a therapeutic response in AML patient samples and animal models of AML that correlates with the suppression of FOXM1 and its transcriptional targets. Addition of low doses of ixazomib increases sensitization of AML cells to chemotherapy backbone drugs cytarabine and the hypomethylator 5-azacitidine. Our results underscore the importance of FOXM1 in AML progression and treatment, and they suggest that targeting it may have therapeutic benefit in combination with standard AML therapies.
Authors
Irum Khan, Marianna Halasi, Anand Patel, Rachael Schultz, Nandini Kalakota, Yi-Hua Chen, Nathan Aardsma, Li Liu, John D. Crispino, Nadim Mahmud, Olga Frankfurt, Andrei L. Gartel
Abstract
BACKGROUND. The red cell distribution width (RDW) is associated with health outcomes. Whether non-RDW risk information is contained in RBC sizes is unknown. This study evaluated the association of the percentage of extreme macrocytic RBCs (%Macro, RBC volume > 120 fl) and microcytic RBCs (%Micro, RBC volume < 60 fl) and the RDW–size distribution (RDW-sd) with mortality and morbidity. METHODS. Patients (females, n = 165,770; males, n = 100,210) at Intermountain Healthcare were studied if they had a hematology panel between May 2014 and September 2016. Adjusted sex-specific associations of %Macro/%Micro and RDW-sd with mortality and 33 morbidities were evaluated. RESULTS. Among females with fourth-quartile values of %Macro quartile and %Micro (referred to throughout as 4/4), there was an average of 7.2 morbidities versus 2.9 in the lowest risk (LR1) categories, 1/1, 1/2, 2/1, and 2/2 (P < 0.001). Among males, those in the 4/4 category had 8.0 morbidities, while those in the LR1 had 3.4 (P < 0.001). Cox regressions found %Macro/%Micro (4/4 vs. LR1, females: hazard ratio [HR] = 1.97 [95% CI = 1.53, 2.54]; males: HR = 2.17 [CI = 1.72, 2.73]), RDW-sd (quartile 4 vs. 1, females: HR = 1.33 [CI = 1.04, 1.69]; males: HR = 1.41 [CI = 1.10, 1.80]), and RDW (quartile 4 vs. 1, females: HR = 1.59 [CI = 1.26, 2.00]; males: HR = 1.23 [CI = 0.99, 1.52]) independently predicted mortality. Limitations include that the observational design did not reveal causality and unknown confounders may be unmeasured. CONCLUSIONS. Concomitantly elevated %Macro and %Micro predicted the highest mortality risk and the greatest number of morbidities, revealing predictive ability of RBC volume beyond what is measured clinically. Mechanistic investigations are needed to explain the biological basis of these observations. FUNDING. This study was supported by internal Intermountain Heart Institute funds and in-kind support from Sysmex America Inc.
Authors
Benjamin D. Horne, Joseph B. Muhlestein, Sterling T. Bennett, Joseph Boone Muhlestein, Kurt R. Jensen, Diane Marshall, Tami L. Bair, Heidi T. May, John F. Carlquist, Matthew Hegewald, Stacey Knight, Viet T. Le, T. Jared Bunch, Donald L. Lappé, Jeffrey L. Anderson, Kirk U. Knowlton
Abstract
Germline SAMD9 and SAMD9L mutations cause a spectrum of multisystem disorders that carry a markedly increased risk of developing myeloid malignancies with somatic monosomy 7. Here, we describe 16 siblings, the majority of which were phenotypically normal, from 5 families diagnosed with myelodysplasia and leukemia syndrome with monosomy 7 (MLSM7; OMIM 252270) who primarily had onset of hematologic abnormalities during the first decade of life. Molecular analyses uncovered germline SAMD9L (n = 4) or SAMD9 (n = 1) mutations in these families. Affected individuals had a highly variable clinical course that ranged from mild and transient dyspoietic changes in the bone marrow to a rapid progression of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with monosomy 7. Expression of these gain-of-function SAMD9 and SAMD9L mutations reduces cell cycle progression, and deep sequencing demonstrated selective pressure favoring the outgrowth of clones that have either lost the mutant allele or acquired revertant mutations. The myeloid malignancies of affected siblings acquired cooperating mutations in genes that are also altered in sporadic cases of AML characterized by monosomy 7. These data have implications for understanding how SAMD9 and SAMD9L mutations contribute to myeloid transformation and for recognizing, counseling, and treating affected families.
Authors
Jasmine C. Wong, Victoria Bryant, Tamara Lamprecht, Jing Ma, Michael Walsh, Jason Schwartz, Maria del pilar Alzamora, Charles G. Mullighan, Mignon L. Loh, Raul Ribeiro, James R. Downing, William L. Carroll, Jeffrey Davis, Stuart Gold, Paul C. Rogers, Sara Israels, Rochelle Yanofsky, Kevin Shannon, Jeffery M. Klco
Abstract
Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Krasex3op) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that Kras protein levels were significantly increased in bone marrow (BM) HSCs in Krasex3op/ex3op mice, demonstrating that the translation of Kras in HSCs is normally constrained by rare codons. Krasex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells, and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in Erk1/2 phosphorylation and cyclin-dependent kinase 4 and 6 (Cdk4/6) activation. Furthermore, both the enhanced colony-forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced expansion of HSCs, progenitor cells, and B cells in a hematopoietic cell–autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence, which may speak to the importance of codon usage in mammalian biology.
Authors
Joshua P. Sasine, Heather A. Himburg, Christina M. Termini, Martina Roos, Evelyn Tran, Liman Zhao, Jenny Kan, Michelle Li, Yurun Zhang, Stéphanie C. de Barros, Dinesh S. Rao, Christopher M. Counter, John P. Chute
Abstract
Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.
Authors
Charlotte F. Inman, Suzy A. Eldershaw, Joanne E. Croudace, Nathaniel J. Davies, Archana Sharma-Oates, Tanuja Rai, Hayden Pearce, Mirjana Sirovica, Y.L. Tracey Chan, Kriti Verma, Jianmin Zuo, Sandeep Nagra, Francesca Kinsella, Jane Nunnick, Rasoul Amel-Kashipaz, Charles Craddock, Ram Malladi, Paul Moss
Abstract
In response to collagen stimulation, platelets use a coordinated system of fluid entry to undergo membrane ballooning, procoagulant spreading, and microvesiculation. We hypothesized that water entry was mediated by the water channel aquaporin-1 (AQP1) and aimed to determine its role in the platelet procoagulant response and thrombosis. We established that human and mouse platelets express AQP1 and localize to internal tubular membrane structures. However, deletion of AQP1 had minimal effects on collagen-induced platelet granule secretion, aggregation, or membrane ballooning. Conversely, procoagulant spreading, microvesiculation, phosphatidylserine exposure, and clot formation time were significantly diminished. Furthermore, in vivo thrombus formation after FeCl3 injury to carotid arteries was also markedly suppressed in AQP1-null mice, but hemostasis after tail bleeding remained normal. The mechanism involves an AQP1-mediated rapid membrane stretching during procoagulant spreading but not ballooning, leading to calcium entry through mechanosensitive cation channels and a full procoagulant response. We conclude that AQP1 is a major regulator of the platelet procoagulant response, able to modulate coagulation after injury or pathologic stimuli without affecting other platelet functional responses or normal hemostasis. Clinically effective AQP1 inhibitors may therefore represent a novel class of antiprocoagulant antithrombotics.
Authors
Ejaife O. Agbani, Christopher M. Williams, Yong Li, Marion T.J. van den Bosch, Samantha F. Moore, Adele Mauroux, Lorna Hodgson, Alan S. Verkman, Ingeborg Hers, Alastair W. Poole
Abstract
BACKGROUND. There are very few studies investigating metabolic biomarkers to predict acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Metabolic models can provide a framework for analyzing the information-rich omics data sets in this setting. METHODS. Four hundred and fifty-six samples from one hundred and fourteen consecutive patients who underwent HSCT from January 2012 to May 2014 were collected for this study. The changes in serum metabolite levels were investigated using a gas chromatography–mass spectrometry–based metabolomics approach and underwent statistical analysis. RESULTS. Significant metabolic changes were observed on day 7. The stearic acid/palmitic acid (SA/PA) ratio was effective in the diagnosis of grade II–IV aGVHD. Multivariate analysis showed that patients with high SA/PA ratios on day 7 after HSCT were less likely to develop II–IV aGVHD than patients with low SA/PA ratios (odds ratio [OR] = 0.06, 95% CI 0.02–0.18, P < 0.001). After the adjustment for clinical characteristics, the SA/PA ratio had no significant effect on overall survival (hazard ratio [HR] = 1.95, 95% CI 0.92–4.14, P = 0.08), and patients in the high SA/PA ratio group were significantly more likely to relapse than those in the low ratio group (HR = 2.26, 95% CI 1.04–4.91, P = 0.04). CONCLUSION. Our findings suggest that the SA/PA ratio on day 7 after HSCT is an excellent biomarker to predict both aGVHD and relapse. The serum SA/PA ratio measured on day 7 after transplantation may improve risk stratification for aGVHD and relapse after allogeneic stem cell transplantation. FUNDING. National Natural Science Foundation of China (81470346, 81773361), Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Natural Science Foundation (BK20161204), Innovation Capability Development Project of Jiangsu Province (BM2015004), Jiangsu Medical Junior Talent Person award (QNRC2016707), and NIH (AI129582 and NS106170).
Authors
Xiaojin Wu, Yiyu Xie, Chang Wang, Yue Han, Xiebing Bao, Shoubao Ma, Ahmet Yilmaz, Bingyu Yang, Yuhan Ji, Jinge Xu, Hong Liu, Suning Chen, Jianying Zhang, Jianhua Yu, Depei Wu
Abstract
BACKGROUND. Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring. METHODS. We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples. RESULTS. Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38– AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells. CONCLUSION. The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results. FUNDING. National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children’s Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.
Authors
Elaine Coustan-Smith, Guangchun Song, Sheila Shurtleff, Allen Eng-Juh Yeoh, Wee Joo Chng, Siew Peng Chen, Jeffrey E. Rubnitz, Ching-Hon Pui, James R. Downing, Dario Campana
Abstract
BACKGROUND. Multiple myeloma is usually fatal due to serial relapses that become progressively refractory to therapy. CD19 is typically absent on the dominant multiple myeloma cell population but may be present on minor subsets with unique myeloma-propagating properties. To target myeloma-propagating cells, we clinically evaluated autologous T cells transduced with a chimeric antigen receptor (CAR) against CD19 (CTL019). METHODS. Subjects received CTL019 following salvage high-dose melphalan and autologous stem cell transplantation (ASCT). All subjects had relapsed/refractory multiple myeloma and had previously undergone ASCT with less than 1 year progression-free survival (PFS). RESULTS. ASCT + CTL019 was safe and feasible, with most toxicity attributable to ASCT and no severe cytokine release syndrome. Two of 10 subjects exhibited significantly longer PFS after ASCT + CTL019 compared with prior ASCT (479 vs. 181 days; 249 vs. 127 days). Correlates of favorable clinical outcome included peak CTL019 frequency in bone marrow and emergence of humoral and cellular immune responses against the stem-cell antigen Sox2. Ex vivo treatment of primary myeloma samples with a combination of CTL019 and CAR T cells against the plasma cell antigen BCMA reliably inhibited myeloma colony formation in vitro, whereas treatment with either CAR alone inhibited colony formation inconsistently. CONCLUSION. CTL019 may improve duration of response to standard multiple myeloma therapies by targeting and precipitating secondary immune responses against myeloma-propagating cells. TRIAL REGISTRATION. Clinicaltrials.gov identifier NCT02135406. FUNDING. Novartis, NIH, Conquer Cancer Foundation.
Authors
Alfred L. Garfall, Edward A. Stadtmauer, Wei-Ting Hwang, Simon F. Lacey, Jan Joseph Melenhorst, Maria Krevvata, Martin P. Carroll, William H. Matsui, Qiuju Wang, Madhav V. Dhodapkar, Kavita Dhodapkar, Rituparna Das, Dan T. Vogl, Brendan M. Weiss, Adam D. Cohen, Patricia A. Mangan, Emily C. Ayers, Selene Nunez-Cruz, Irina Kulikovskaya, Megan M. Davis, Anne Lamontagne, Karen Dengel, Naseem D.S. Kerr, Regina M. Young, Donald L. Siegel, Bruce L. Levine, Michael C. Milone, Marcela V. Maus, Carl H. June
Abstract
Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell–derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.
Authors
David A. Slatter, Charles L. Percy, Keith Allen-Redpath, Joshua M. Gajsiewicz, Nick J. Brooks, Aled Clayton, Victoria J. Tyrrell, Marcela Rosas, Sarah N. Lauder, Andrew Watson, Maria Dul, Yoel Garcia-Diaz, Maceler Aldrovandi, Meike Heurich, Judith Hall, James H. Morrissey, Sebastien Lacroix-Desmazes, Sandrine Delignat, P. Vincent Jenkins, Peter W. Collins, Valerie B. O’Donnell
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