Ratio of stemness to interferon signalling as a biomarker and therapeutic target of myeloproliferative neoplasm progression to acute myeloid leukaemia

Progression to aggressive secondary acute myeloid leukaemia (sAML) poses a significant challenge in the management of myeloproliferative neoplasms (MPNs). Since the physiopathology of MPN is closely linked to the activation of interferon (IFN) signalling and that AML initiation and aggressiveness is driven by leukaemia stem cells (LSCs), we investigated these pathways in MPN to sAML progression. We found that high IFN signalling correlated with low LSC signalling in MPN and AML samples, while MPN progression and AML transformation were characterized by decreased IFN signalling and increased LSC signature. A high LSC to IFN expression ratio in MPN patients was associated with adverse clinical prognosis and higher colony forming potential. Moreover, treatment with hypomethylating agents (HMAs) activates the IFN signalling pathway in MPN cells by inducing a viral mimicry response. This response is characterized by double-stranded RNA (dsRNA) formation and MDA5/RIG-I activation. The HMA-induced IFN response leads to a reduction in LSC signature, resulting in decreased stemness. These findings reveal the frequent evasion of viral mimicry during MPN-to-sAML progression, establish the LSC-to-IFN expression ratio as a progression biomarker, and suggests that HMAs treatment can lead to haematological response in murine models by re-activating dsRNA-associated IFN signalling

Hematopoietic stem cells regulation mediated by natural killer cells in a myeloproliferative neoplasm model

A mutação JAK2V617F é o principal defeito molecular observado em neoplasias mieloproliferativas (NMP) negativas para o rearranjo t(9;22)/BCR-ABL1, está presente em 95% dos pacientes com policitemia vera (PV) e é observada em aproximadamente 50% dos pacientes com trombocitemia essencial (TE) e mielofibrose primaria (MFP). Além das mutações, diferentes células do nicho da medula óssea podem contribuir para o início e manutenção da doença. As células Natural Killer (NK) são linfócitos que tem um papel central na resposta imune inata contra tumores. Além das suas propriedades anti-leucêmicas, essas células apresentam uma alta capacidade regulatória que as tornam capazes de reduzir o potencial de enxertia e auto renovação de células-tronco hematopoiéticas (CTH) normais. Entretanto, em neoplasias originadas de transformação neoplásica das CTH, não se sabe até o momento se esta regulação é eficiente. Considerando que as CTH normais podem responder a estímulos mediados pelas células NK, é possível que uma deficiência funcional das células NK contribua para expansão de células-tronco neoplásicas ou que as CTH transformadas se tornem resistentes à ação antileucêmicas das células NK. Para estudar tal hipótese, utilizamos um modelo murino condicional knockin de expressão heterozigótica da mutação Jak2V617F (Jak2VF), no qual os camundongos que expressam a mutação desenvolvem uma neoplasia mieloproliferativa letal que se assemelha à PV humana. Realizamos análises imunofenotípicas e funcionais das células NK dos camundongos Jak2WT e Jak2VF isoladamente e em co-cultivo com CTH, avaliando seu potencial de proliferação, diferenciação e auto renovação. Os resultados mostraram que o baço dos animais doentes apresentava células NK CD27+ e uma redução de células NK CD11b+. Embora não significativo, quando comparados aos animais saudáveis, houve uma tendência ao acumulo de células NK CD27-CD11b- nos camundongos JAK2VF, o que corrobora com o aumento de células imaturas no baço. Além disso, observou-se que o perfil fenotípico das células NK na medula óssea é diferente da população de células NK do baço, na qual, os grupos JAK2WT e JAK2VF apresentou um acumulo de células imaturas e secretórias. Os ensaios funcionais revelaram que, quando comparadas ao grupo controle, as células NK no grupo JAK2VF apresentaram menor capacidade proliferativa e reduziram a frequência de células CD69+ após estimulo com PMA e ionomicina. Além disso, os dados demonstraram que a proporção de células NK CD69+CD107a+ foi similar em ambos os grupos. Os estudos da interação de células NK com CTH por meio do ensaio de CFU, revelou que o contato prévio com células NK isoladas dos animais saudáveis foi capaz de reduzir o potencial clonogênico de CTH, no entanto, o mesmo não foi observado com as CTH foram expostas a interação com células NK JAK2VF. Em resumo, os resultados obtidos neste estudo permitiram caracterizar um perfil imaturo e secretório de células NK JAK2VF. Funcionalmente, os dados revelaram que essas células apresentam menor capacidade de ativação, proliferação e de inibição do potencial clonogênico CTH. O comportamento fenotípico e funcional aberrante das células NK JAK2VF sugere um comprometimento da imunovigilancia do câncer e pode associar-se com a sustentação da doença. Além disso os dados apresentados forneceram uma nova perspectiva sobre comportamento das células NK no contexto de neoplasias mieloides. Acreditamos que a compreensão da heterogeneidade fenotípica e funcional dessas células possa contribuir para o desenvolvimento de novas estratégias terapêuticas direcionadas ao controle das funções das CTH transformadas.JAK2V617F mutation is the most common molecular abnormality observed in t(9;22)/BCR-ABL1- -negative myeloproliferative neoplasms (MPN). It is present in 95% of patients with Polycythemia Vera (PV) and observed in approximately 50% of patients with Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). In addition to driver mutations that activate the JAK/STAT signaling pathway, different cells from the bone marrow niche may contribute to the initiation and maintenance of the disease. Natural Killer (NK) cells are a subset of lymphocytes that play a central role in the innate immune response to tumors. Besides their anti-leukemic properties, these cells have a high regulatory capacity that makes them capable of reducing hematopoietic stem cells (HSCs) potencial of engraftment and self-renewal. However, it is currently unknown if this NK regulation also occurs in the context of neoplastic transformation. Considering that normal HSCs may respond to NK cell-mediated stimuli, it is possible that NK regulation may favor the malignant transformation of HSCs or that neoplastic HSCs are resistant to the its anti-leukemic actions. To address this hypothesis, we used a conditional knockin Jak2V617F mutation (Jak2VF) murine model, which faithfully resembles the main clinical and laboratorial characteristics of human PV. We performed phenotypical and functional analyses of NK cells. Furthermore, co-cultures of Jak2WT and Jak2VFisolated NK cells with wild-type HSCs were used to assess their potential for proliferation, differentiation and self-renewal after NK exposure. In this study, we showed that the frequency of phenotypic profile of NK cells in the spleen of mice that harbor JAK2VF mutation increased CD27+NK cells and reduce the NK CD11b+ cells profile. Although we did not reach a statistical significance, when compared to healthy mice, there was a tendency to accumulate NK CD27-CD11b- cells in JAK2VF mice, which corroborates the increase in immature cells in the spleen. In addition, the bone marrow-derivate NK cells presented a similar phenotype of NK cells from the spleen of JAK2VF mice. Functional assays showed that, when compared to the control group, NK cells in the JAK2VF group presented reduction of their proliferative capacity and decrased the frequency of CD69+ cells after stimulation with PMA and ionomycin. Furthemore, the data demonstrated in both group a similar proportion of CD69+CD107a+NK cells after stimulation with PMA plus ionomycin. The evaluation of interaction between NK cells and HSC through the CFU assay revealed that previous contact with NK cells isolated from healthy animals was able to reduce the clonogenic potential of HSC, however, the same was not observed with HSCs exposed to interaction with NK JAK2VF cells. In summary, the results obtained in this study allowed to characterize an immature and secretory profile of NK JAK2VF cells. Functionally, the data revealed that these cells have less capacity for activation, proliferation and inhibition of HSC clonogenic potential. The arrest of immature profile and defects of functional NK JAK2VF cells suggests an impairment of cancer immunovigilance. Furthermore, the data provided a new perspective on the behavior of NK cells in the context of myeloid malignances. We believe that understanding the phenotypic and functional heterogeneity of these cells can contribute to the development of new therapeutic strategies aimed at controlling the functions of transformed HSCs. Therefore, identifying the mechanisms of NK-cell deficiency in myeloid neoplasms and the detailed interaction between these cells and the malignant stem cell may help to establish combination therapeutic strategies that may improve therapy responses

DataSheet_1_Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.pdf

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.</p