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Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the myelodysplastic/myeloproliferative syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S, and G245V substitutions. We previously showed that ETNK1 mutations cause a decreased catalytic activity of the enzyme. Despite this evidence however, their oncogenic role remained largely unexplained. Since ETNK1 activity is essential for the synthesis of phosphatidylethanolamine (PE) and given that PE is one of the most abundant phospholipids in the inner membrane of mitochondria, we focused our attention on mitochondrial activity. In order to characterize the oncogenic effect of ETNK1 variants we generated CRISPR/Cas9 clones carrying heterozygous N244S mutation and homozygous ETNK1 deletion (KO cells) on the 293 Flp-In™ cell-line. Both N244S and KO cells showed a significant increase in mitochondrial activity (1.78 and 2.13 fold increase, respectively; p= 0.0096 and p=0.0050) compared to WT, as assessed by MitoTracker™ Red. In line with this finding, electron microscopy revealed a significant modification in mitochondria morphology for N244S and KO cells, changing from an elongated, tubular shape to a round, swollen one. ATP (1.67 and 1.68 fold; p

Local skin reactions to subcutaneous injections of interferon beta (IFNB) or glatiramer acetate (GA) in multiple sclerosis (MS) are frequent, while severe cutaneous toxicity is rare. Both IFNB and GA are immunomodulatory drugs that have excellent safety profiles and are currently used for treatment of MS. They are administered by SC injection every other day for IFNB-1b, three times a week for IFNB-1a or daily for 20 mg for GA. The most common adverse effects, which occur in approximately 20-60% of patients, include pain, inflammation and induration at the injection sites. Another adverse effect is frank panniculitis followed by localized lipoatrophy at the injection sites, which has been described in half of the patients receiving GA injections but is also described with Subcutaneous IFNB-1b. No guidelines have yet been established for the treatment of skin reactions, which is a frequent point for discussion between neurologists and dermatologists. In addition, no treatment has been found for established lipoatrophy. The prevention and management of cutaneous side-effects include patient education, regular examination and manual palpation of all injection sites. Non-steroid antiinflammatory gels, local corticosteroids or endermology can help patients to resolve side-effects and to continue immunomodulatory treatment.

Il gene di fusione ETV6::RUNX1 (E/R), generato durante la vita fetale dalla t(12;21), è l’alterazione più frequente nella leucemia linfoblastica acuta infantile (LLA). Tuttavia, E/R non è sufficiente per determinare l’insorgenza della malattia, ma ulteriori mutazioni sono necessarie per causare la leucemia nell’1% dei casi E/R +. Si ritiene che la causa della malattia sia da attribuire ad una risposta infiammatoria e immunitaria aberrante nei confronti di patogeni comuni, la quale può guidare la trasformazione maligna delle cellule pre-leucemiche E/R+, tramite l’acquisizione di mutazioni secondarie. Pertanto, l’obiettivo di questo lavoro è stato quello di meglio comprendere il ruolo delle infezioni/infiammazioni, nel contesto della nicchia ematopoietica, nella patogenesi della malattia. Per fare ciò, ci siamo avvalsi dell’utilizzo di due modelli uno in vitro ed uno in vivo. Il modello in vitro di nicchia mesenchimale competitiva è stato utilizzato per studiare le caratteristiche delle cellule pre-leucemiche E/R+ e il crosstalk con il microambiente midollare. Il modello è stato ottenuto co-coltivando la linea cellulare proB murina Ba/F3 esprimente E/R e cellule Ba/F3 controllo (in un rapporto di 80:20) con cellule stromali mesenchimali isolate da midollo (BM-MSC). Le condizioni infiammatorie sono state riprodotte stimolando il modello con citochine pro-infiammatorie IL6/IL1β/TNFα. In seguito a stimolazione, abbiamo osservato che le BM-MSC attraggono fortemente le cellule Ba/F3 E/R+ tramite l’asse CXCL1-CXCR2. In aggiunta, nel modello di nicchia midollare infiammata, abbiamo osservato un aumento del danno al DNA sia nelle cellule controllo che nelle cellule E/R+, ma mentre le prime vanno incontro ad apoptosi, le seconde sopravvivono e possono così subire una trasformazione tumorale. (Beneforti L, et al. 2020). Per confermare i risultati ottenuti in vitro, ci siamo avvalsi di un modello di topo transgenico Sca1- E/R. Questi topi spostati da uno stabulario definito specific-pathogen-free (SPF) a uno convenzionale (CF) sviluppano la B-ALL a bassa penetranza, simulando così la condizione umana. Al fine di studiare l’impatto dell’infezione a breve e lungo termine sul compartimento ematopoietico progenitore (Lin-Sca1+c-Kit+ (LSK)) e staminale (LSK CD48-CD150+) abbiamo determinato la loro frequenza nel midollo osseo (BM) dei topi stabulati per 4 e 12 settimane nella CF. In seguito a spostamento dei topi nella CF, abbiamo osservato che mentre l’esposizione a breve termine non influenza il numero delle cellule progenitrici e staminali, l’esposizione a lungo termine porta a una forte espansione del solo compartimento staminale, esclusivamente nei topi Sca1-E/R. Tale espansione era preceduta da un aumento di cellule progenitrici E/R+ nel sangue periferico (PB) con un picco tra 4 e 8 settimane di stabulazione nella CF. Le cellule mobilizzate avevano anche livelli di ROS maggiori rispetto al BM, specialmente quelle pre-leucemiche, che possono renderle più suscettibili a sviluppare altre mutazioni. Inoltre, le cellule E/R+ hanno mostrato una risposta chemiotattica ridotta verso CXCL12, chemochina importante nella ritenzione delle cellule immature nel BM. Infine, è stato valutato il profilo citochinico del BM e PB dei topi stabulati nella SPF e nella CF. Tra le 32 citochine analizzate, abbiamo osservato l’up-regolazione di IL9, G-CSF e IL1 nel siero dei topi WT stabulati nella CF rispetto ai topi Sca1-E/R, mentre nel BM non è stata riscontrata alcuna differenza. I risultati ottenuti sia in vitro che in vivo mostrano come l’infezione/infiammazione possa produrre dei cambiamenti nelle cellule pre-leucemiche E/R e renderle più attive e più prone all’acquisizione di ulteriori lesioni genetiche, ma ulteriori studi, specialmente con il modello in vivo, saranno necessari per comprendere il ruolo dell’infezione/infiammazione nel mantenimento della fase pre-leucemica e l’eventuale evoluzione maligna. ETV6::RUNX1(E/R) fusion gene, arising in utero from t(12;21), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, ETV6::RUNX1 is insufficient to overt disease and additional genetic lesions can trigger leukemia development in 1% of ETV6::RUNX1 cases. Dysregulated inflammatory and immune response to common infection is thought to be the major player in ETV6::RUNX1 malignant transformation, driving the acquisition of secondary mutations. Thus, we aim to comprehend the role of infection(s)/inflammation, in the hematopoietic niche, on the pathogenesis of the disease. In particular, we took advantage of both in vitro and in vivo models. We set up an in vitro model of competitive mesenchymal niche to study E/R+ pre-leukemic cells and BM microenvironment crosstalk. We co-cultured BaF3 proB cell line expressing E/R and control Ba/F3 (ratio of 80:20%) on BM-MSCs. We reproduced inflammatory conditions by treating competitive niche model with IL6/IL1β/TNFα pro-inflammatory cytokines. Following cytokines stimulation, we found that BM-MSCs strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. in addition, inflamed niche model lead DNA damage in both populations but while control cells undergo apoptosis, E/R+ Ba/F3 survive and can develop additionally genetic lesions (Beneforti L, et al. 2020). To confirm our in vitro results, we took advantage of a Sca1-ETV6-RUNX1 transgenic mouse model. These mice shifted from a specific pathogen free facility (SPF) to the conventional facility (CF) can develop B-ALL at low penetrance, thus resembling the human condition (Rodríguez-Hernandez G. et al. 2017). In order to explore the short- and the long-term impact of the infection on the immature compartment, we firstly determined the frequency of HSPCs (Lin- Sca1+c-Kit+ (LSK)) and long-term HSCs (LT-HSCs: LSK CD48-CD150+) in the bone marrow (BM) of mice housed in the CF for 4 and 12 weeks. After shifting the mice to the CF, we observed that while the short-term exposure did not affect both HSPCs and LT-HSCs frequency, the long-term exposure induced a sharp increase only in the LT-HSCs compartment of Sca1-ETV6-RUNX1 mice. Moreover, when the mice were exposed to infection, we found that ETV6::RUNX1 HSPCs egress to the peripheral blood (PB) reaching the highest peak between 4 and 8 weeks. Circulating cells showed higher ROS levels compared to BM cells, especially of pre-leukemic cells which in turn can lead to the acquisition of additional genetic lesions. Moreover, ETV6::RUNX1 pre-leukemic cells showed altered chemotactic response toward CXCL12, a chemokine involved in the retention of immature cells in the BM. Finally, we assessed the cytokines profile of BM and PB of mice housed in SPF and CF. Among the 32 cytokines analyzed, we found the up-regulation of IL9, G-CSF and IL1 in the PB serum only in WT mice moved to CF, while in the BM we did not find any cytokines differentially secreted. Taken together both in vitro and in vivo results showed that infection/inflammation can modify ETV6::RUNX1 pre-leukemic cells behavior and eventually guide the acquisition of further genetic lesions, but further studies, especially with the in vivo model, are essential to understand the role of infection/inflammation in the maintenance of the covert disease as well as in stimulating the molecular changes that lead to leukemia onset.

ETNK1 kinase is responsible for the phosphorylation of ethanolamine to phosphoethanolamine (P-Et) (Kennedy, 1956, J Biol Chem). Recurrent somatic mutations occurring on ETNK1 were identified in about 13% of patients affected by atypical chronic myeloid leukemia (aCML), in 3-14% of chronic myelomonocytic leukemia (CMML), and in 20% of systemic mastocytosis (SM) patients with eosinophilia (Gambacorti-Passerini, 2015, Blood; Lasho, 2015, Blood Cancer J). ETNK1 mutations, encoding for H243Y, N244S/T/K, and G245V/A amino acid substitutions, cluster in a very narrow region of the ETNK1 catalytic domain and cause an impairment of ETNK1 enzymatic activity leading to a significant decrease in the intracellular concentration of P-Et (Gambacorti-Passerini, 2015, Blood). Despite this evidence, however, their oncogenic role remained largely unexplained. Here, we investigated the specific role of these mutations by using cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients' samples. We showed that mutated ETNK1 causes a significant increase in mitochondrial activity (1.87 fold increase compared to WT; p=0.0002) and in ROS production (2.05 fold increase compared to WT; p Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype., ETNK1 mutations are recurrent in leukemia but how they contribute to oncogenesis is still unclear. Here, the authors show that ETNK1 mutations increase mitochondrial activity, ROS and H2AX levels and that these effects can be rescued upon phosphoethanolamine supplementation.