Inferring the dynamic of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells (HSC) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon alpha (IFNα) has demonstrated some efficacy in inducing molecular remission in MPN. In order to determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in MPN patients by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured several times per year the clonal architecture of early and late hematopoietic progenitors (84,845 measurements) and the global variant allele frequency in mature cells (409 measurements). Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSC. Our data support the hypothesis that IFNα targets JAK2V617F HSC by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSC and increases with high IFNα dosage in heterozygous JAK2V617F HSC. Besides, we found that the molecular responses of CALRm HSC to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and high dosage of IFNα correlates with worse outcomes. Together, our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dosage.

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Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

PLoS ONE ◽

10.1371/journal.pone.0247858 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0247858

Author(s):

Ron Baik ◽

Stacia K. Wyman ◽

Shaheen Kabir ◽

Jacob E. Corn

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Cytokine Signaling ◽

Competitive Growth ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Myeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop “scarless” Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

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Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

10.1101/855452 ◽

2019 ◽

Author(s):

Ron Baik ◽

Stacia K. Wyman ◽

Shaheen Kabir ◽

Jacob E. Corn

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Cytokine Signaling ◽

Competitive Growth ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

AbstractMyeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop “scarless” Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

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Hematopoietic fitness of JAK2V617F Myeloproliferative Neoplasms is linked to clinical outcome

10.1101/2021.01.28.21250575 ◽

2021 ◽

Author(s):

Ghaith Abu-Zeinah ◽

Silvana Di Giandomenico ◽

Tatiana Cruz ◽

Elwood Taylor ◽

Ellen K Ritchie ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Treatment Options ◽

Principal Component ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Disease Modifying ◽

Serial Samples ◽

Fitness Levels

AbstractMyeloproliferative Neoplasms (MPN) harbor highly recurrent driver mutations affecting targetable kinases yet treatment options for these phenotypically diverse diseases are limited, and patients experience significant morbidity and shortened survival. The most important disease-related complications—thrombosis, transformation and death—are not used as clinical trial endpoints due to the long follow-up required to assess such disease modifying activity. A reliable monitoring biomarker linking MPN biology with these important clinical outcomes is missing. MPN driver mutation allele frequency (MAF) from whole blood or marrow (WB) does not faithfully predict MPN phenotype, clinical progression or response. This is likely because WB MAF is a composite measure of alleles from a heterogenous and variable mixture of mature leukocytes and, as such, does not report any information about the critical MPN stem and progenitor cells (MPN-SPCs). Driver mutations allow MPN cells to outcompete their normal hematopoietic counterparts and this competitive advantage—increased “fitness”—underlies core biology of MPN pathogenesis. We developed an approach to directly measure MPN fitness from samples. We measured fitness in 115 samples from 84 patients with JAK2V617F MPNs by quantifying MAF of 11 well-defined and strictly validated hematopoietic stem, progenitor and mature cell populations purified from routinely collected blood and marrow specimens. Unsupervised, hierarchical clustering of MPN fitness revealed 4 major fitness levels: F1, F2, F3, and F4 with significantly different but overlapping clinical features and diagnoses. Notably, these four fitness levels were associated with significantly different event-free survival (EFS): 95% (F1), 81% (F2), 73% (F3), 50% (F4) at 24 months (log-rank p=0.017). In contrast, WB MAF quartile failed to predict EFS. Multivariable models showed that fitness was associated with event risk independent of age, sex, duration of disease, MPN diagnosis and WB MAF. Principal component analysis allowed convenient projection of the 11-component MAF fitness measures to reduce dimensionality and develop a model for relative risk (RR) of event that could be used to assess individual or serial samples. Serial samples with more than a year of follow-up was available for 13 patients. We found that a reduction of this RR score was associated with a therapeutic response (p=0.045). In contrast, increasing RR overtime portended a disease-related event (p=0.045). Changes in WB MAF did not correlate with RR (r2=0.022) possibly explaining why WB MAF failed to predict events. These data demonstrate that fitness dynamics from serial blood samples can be used as a monitoring biomarker to assess changes in RR over time. Thus, fitness risk is a promising endpoint alongside corresponding clinical parameters such as blood counts, spleen size and marrow fibrosis grade. Our study offers a feasible approach to monitor the MPN biology central to disease progression and can be used in clinical trials to efficiently identify disease-modifying, potentially life-prolonging treatments.

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The Thrombopoietin Receptor, MPL, Is a Therapeutic Target of Opportunity in the MPN

Frontiers in Oncology ◽

10.3389/fonc.2021.641613 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jerry L. Spivak ◽

Alison R. Moliterno

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Therapeutic Target ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Thrombopoietin Receptor

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis share driver mutations that either activate the thrombopoietin receptor, MPL, or indirectly activate it through mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, although the myeloproliferative neoplasms are classified as neoplasms because they are clonal hematopoietic stem cell disorders, the mutations affecting MPL or JAK2 are gain-of-function, resulting in increased production of normal erythrocytes, myeloid cells and platelets. Constitutive JAK2 activation provides the basis for the shared clinical features of the myeloproliferative neoplasms. A second molecular abnormality, impaired posttranslational processing of MPL is also shared by these disorders but has not received the recognition it deserves. This abnormality is important because MPL is the only hematopoietic growth factor receptor expressed in hematopoietic stem cells; because MPL is a proto-oncogene; because impaired MPL processing results in chronic elevation of plasma thrombopoietin, and since these diseases involve normal hematopoietic stem cells, they have proven resistant to therapies used in other myeloid neoplasms. We hypothesize that MPL offers a selective therapeutic target in the myeloproliferative neoplasms since impaired MPL processing is unique to the involved stem cells, while MPL is required for hematopoietic stem cell survival and quiescent in their bone marrow niches. In this review, we will discuss myeloproliferative neoplasm hematopoietic stem cell pathophysiology in the context of the behavior of MPL and its ligand thrombopoietin and the ability of thrombopoietin gene deletion to abrogate the disease phenotype in vivo in a JAK2 V617 transgenic mouse model of PV.

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Myelodysplastic Syndromes in the Postgenomic Era and Future Perspectives for Precision Medicine

Cancers ◽

10.3390/cancers13133296 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3296

Author(s):

Ioannis Chanias ◽

Kristina Stojkov ◽

Gregor Stehle ◽

Michael Daskalakis ◽

Helena Simeunovic ◽

...

Keyword(s):

Precision Medicine ◽

Myelodysplastic Syndromes ◽

Clonal Evolution ◽

Driver Mutations ◽

Future Perspectives ◽

Hematopoietic Stem ◽

Secondary Acute Myeloid Leukemia ◽

Stem And Progenitor Cells ◽

Unfit Patients ◽

Generation Sequencing

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders caused by sequential accumulation of somatic driver mutations in hematopoietic stem and progenitor cells (HSPCs). MDS is characterized by ineffective hematopoiesis with cytopenia, dysplasia, inflammation, and a variable risk of transformation into secondary acute myeloid leukemia. The advent of next-generation sequencing has revolutionized our understanding of the genetic basis of the disease. Nevertheless, the biology of clonal evolution remains poorly understood, and the stochastic genetic drift with sequential accumulation of genetic hits in HSPCs is individual, highly dynamic and hardly predictable. These continuously moving genetic targets pose substantial challenges for the implementation of precision medicine, which aims to maximize efficacy with minimal toxicity of treatments. In the current postgenomic era, allogeneic hematopoietic stem cell transplantation remains the only curative option for younger and fit MDS patients. For all unfit patients, regeneration of HSPCs stays out of reach and all available therapies remain palliative, which will eventually lead to refractoriness and progression. In this review, we summarize the recent advances in our understanding of MDS pathophysiology and its impact on diagnosis, risk-assessment and disease monitoring. Moreover, we present ongoing clinical trials with targeting compounds and highlight future perspectives for precision medicine.

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Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

Stem Cells Translational Medicine ◽

10.5966/sctm.2016-0240 ◽

2016 ◽

Vol 6 (3) ◽

pp. 864-876 ◽

Cited By ~ 18

Author(s):

Jennifer L. Gori ◽

Jason M. Butler ◽

Balvir Kunar ◽

Michael G. Poulos ◽

Michael Ginsberg ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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A Case of Myeloproliferative Neoplasm with BCR-FGFR1 Rearrangement: Favorable Outcome after Haploidentical Allogeneic Transplantation

Case Reports in Hematology ◽

10.1155/2018/5724960 ◽

2018 ◽

Vol 2018 ◽

pp. 1-4 ◽

Cited By ~ 1

Author(s):

Paola Villafuerte-Gutiérrez ◽

Montserrat López Rubio ◽

Pilar Herrera ◽

Eva Arranz

Keyword(s):

Kinase Inhibitors ◽

Fusion Gene ◽

Myeloproliferative Neoplasms ◽

Therapeutic Option ◽

Myeloproliferative Neoplasm ◽

Allogeneic Transplantation ◽

Term Survival ◽

Hematopoietic Stem ◽

Health Organization

Hematopoietic myeloproliferative neoplasms with FGFR1 rearrangement result in the 8p11 myeloproliferative syndrome that in the current Word Health Organization classification is designated as “myeloid and lymphoid neoplasm with FGFR1 abnormalities.” We report the case of a 66-year-old man who had clinical features that resembled chronic myeloid leukaemia (CML), but bone marrow cytogenetic and fluorescent in situ hybridization (FISH) studies showed t(8;22)(p11;q11) and BCR-FGFR1 fusion gene. He was initially managed with hydroxyurea, and given the aggressive nature of this disease, four months later, the patient underwent an allogeneic hematopoietic stem-cell transplantation (HSCT) from an HLA-haploidentical relative. Currently, HSCT may be the only therapeutic option for long-term survival at least until more efficacious tyrosine kinase inhibitors (TKIs) become available.

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Loss of Atg2b and Gskip impairs the maintenance of the hematopoietic stem cell pool size

Molecular and Cellular Biology ◽

10.1128/mcb.00024-21 ◽

2021 ◽

Author(s):

Shun-suke Sakai ◽

Atsushi Hasegawa ◽

Ryosuke Ishimura ◽

Naoki Tamura ◽

Shun Kageyama ◽

...

Keyword(s):

Myeloproliferative Neoplasm ◽

Pool Size ◽

Double Knockout ◽

Hematopoietic Stem ◽

Cell Pool ◽

Expression Of Genes ◽

Genes Encoding ◽

Differentiation Efficiency ◽

Stem Cell Pool

A germline copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP , was identified in families with myeloproliferative neoplasm (MPN). Herein, we show that mice lacking both Atg2b and Gskip , but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP , the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double knockout fetal livers were significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

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Current Concepts of Pathogenesis and Treatment of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms

Hämostaseologie ◽

10.1055/a-1447-6667 ◽

2021 ◽

Vol 41 (03) ◽

pp. 197-205

Author(s):

Franziska C. Zeeh ◽

Sara C. Meyer

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mutational Analysis ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Standard Of Care ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Therapeutic Decisions ◽

Jak2 Inhibitors

AbstractPhiladelphia chromosome-negative myeloproliferative neoplasms are hematopoietic stem cell disorders characterized by dysregulated proliferation of mature myeloid blood cells. They can present as polycythemia vera, essential thrombocythemia, or myelofibrosis and are characterized by constitutive activation of JAK2 signaling. They share a propensity for thrombo-hemorrhagic complications and the risk of progression to acute myeloid leukemia. Attention has also been drawn to JAK2 mutant clonal hematopoiesis of indeterminate potential as a possible precursor state of MPN. Insight into the pathogenesis as well as options for the treatment of MPN has increased in the last years thanks to modern sequencing technologies and functional studies. Mutational analysis provides information on the oncogenic driver mutations in JAK2, CALR, or MPL in the majority of MPN patients. In addition, molecular markers enable more detailed prognostication and provide guidance for therapeutic decisions. While JAK2 inhibitors represent a standard of care for MF and resistant/refractory PV, allogeneic hematopoietic stem cell transplantation remains the only therapy with a curative potential in MPN so far but is reserved to a subset of patients. Thus, novel concepts for therapy are an important need, particularly in MF. Novel JAK2 inhibitors, combination therapy approaches with ruxolitinib, as well as therapeutic approaches addressing new molecular targets are in development. Current standards and recent advantages are discussed in this review.

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Synergic Crosstalk between Inflammation, Oxidative Stress, and Genomic Alterations in BCR–ABL-Negative Myeloproliferative Neoplasm

Antioxidants ◽

10.3390/antiox9111037 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1037

Author(s):

Alessandro Allegra ◽

Giovanni Pioggia ◽

Alessandro Tonacci ◽

Marco Casciaro ◽

Caterina Musolino ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cell ◽

Chronic Inflammation ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Tumor Stage ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms ◽

Inflammatory State

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have recently been revealed to be related to chronic inflammation, oxidative stress, and the accumulation of reactive oxygen species. It has been proposed that MPNs represent a human inflammation model for tumor advancement, in which long-lasting inflammation serves as the driving element from early tumor stage (over polycythemia vera) to the later myelofibrotic cancer stage. It has been theorized that the starting event for acquired stem cell alteration may occur after a chronic inflammation stimulus with consequent myelopoietic drive, producing a genetic stem cell insult. When this occurs, the clone itself constantly produces inflammatory components in the bone marrow; these elements further cause clonal expansion. In BCR–ABL1-negative MPNs, the driver mutations include JAK 2, MPL, and CALR. Transcriptomic studies of hematopoietic stem cells from subjects with driver mutations have demonstrated the upregulation of inflammation-related genes capable of provoking the development of an inflammatory state. The possibility of acting on the inflammatory state as a therapeutic approach in MPNs appears promising, in which an intervention operating on the pathways that control the synthesis of cytokines and oxidative stress could be effective in reducing the possibility of leukemic progression and onset of complications.

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