Angela G. Fleischman

Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML

BACKGROUND The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms. METHODS To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies. RESULTS We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib. CONCLUSIONS Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.

The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition

We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease.

ALDH marks leukemia stem cell.

In this issue of Blood,Gerber et al use aldehyde dehydrogenase (ALDH) activity to further subdivide the CD34(+)CD38(-) compartment in the bone marrow of acute myeloid leukemia (AML) patients. They identify a unique population with intermediate ALDH activity (ALDH(int)) that contains leukemia stem cells (LSCs). Moreover, persistence of this population after therapy is a marker of clinically significant minimal residual disease.

BCR-ABL1 promotes leukemia by converting p27 into a cytoplasmic oncoprotein.

Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27(CK-)) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27(T187A)) or nuclear retention (p27(S10A)) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization.

Causal role for JAK2 V617F in thrombosis

In this issue of Blood , Hobbs et al use a JAK2 V617F knock-in mouse model to interrogate the impact of JAK2 V617F on thrombosis and demonstrate altered function of megakaryocytes and platelets in the context of JAK2 V617F expression.[1][1] ![Figure][2] JAK2 V617F induces biological changes

Inflammation as a Driver of Clonal Evolution in Myeloproliferative Neoplasm

Our understanding of inflammations role in the pathogenesis of myeloproliferative neoplasm (MPN) is evolving. The impact of chronic inflammation, a characteristic feature of MPN, likely goes far beyond its role as a driver of constitutional symptoms. An inflammatory response to the neoplastic clone may be responsible for some pathologic aspects of MPN. Moreover, JAK2V617F mutated hematopoietic stem and progenitor cells are resistant to inflammation, and this gives the neoplastic clone a selective advantage allowing for its clonal expansion. Because inflammation plays a central role in MPN inflammation is a logical therapeutic target in MPN.

Hematopoietic stem cell transplantation for myelofibrosis: where are we now?

PURPOSE OF REVIEW A succinct yet comprehensive review of the biology of myeloproliferative neoplasms and therapeutic options with a focus on rational decision making for hematopoietic stem cell transplantation. RECENT FINDINGS The introduction of Janus kinase inhibitors for myelofibrosis have ushered in a new era for treatment of constitutional symptoms and splenomegaly in myelofibrosis, but the effect of these agents on the natural history of the disease has yet to be clearly defined. Reduced intensity transplants have emerged as the preferred option with recent evidence suggesting fludarabine and melphalan as the optimal conditioning regimen. SUMMARY Myelofibrosis is a rare hematologic malignancy with limited curative therapeutic options. Significant advances in our understanding of disease pathogenesis have led to new targets and new therapeutic options are forthcoming. Hematopoietic stem cell transplantation is at present the only treatment with curative intent; however, the selection of patients who are likely to be best served by this procedure is difficult. As myelofibrosis is an extremely rare disease, randomized clinical trials specifically investigating the role of transplantation in myelofibrosis are unlikely to occur, thus current decision making processes are best guided by retrospective analyses from registry databases and single institution experiences.

The Critical Role of Inflammation in the Pathogenesis and Progression of Myeloid Malignancies

Hematopoietic stem cells (HSCs) maintain an organism’s immune system for a lifetime, and derangements in HSC proliferation and differentiation result in hematologic malignancies. Chronic inflammation plays a contributory if not causal role in HSC dysfunction. Inflammation induces HSC exhaustion, which promotes the emergence of mutant clones that may be resistant to an inflammatory microenvironment; this likely promotes the onset of a myeloid hematologic malignancy. Inflammatory cytokines are characteristically high in patients with myeloid malignancies and are linked to disease initiation, symptom burden, disease progression, and worsened prognostic survival. This review will cover our current understanding of the role of inflammation in the initiation, progression, and complications of myeloid hematologic malignancies, drawing from clinical studies as well as murine models. We will also highlight inflammation as a therapeutic target in hematologic malignancies.

Same mutation, different allele

In this issue of Blood , Lambert and colleagues propose a new disease paradigm for one of the CMPNs, ET. They show that in ET, the JAK2 V617F mutation occurs as multiple independent events. 1 Using a common single nucleotide polymorphism located in the JAK2 gene, they demonstrate that the mutation commonly occurs on both alleles in ET. The authors argue that, because of these new findings, the presence of the JAK2 V617F in ET should not be equated with malignant disease.