Identification Of Functional Mutations Of JAK1 In Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia(ALL)is a malignant clonal disorder caused by abnormal development of hematopoietic stem cells or lymphoid progenitor cells.Genetic abnormalities play an important role in the pathogenesis of ALL,which is not only involved in driving the disease,but also related to the relapse,and become the hallmarks of risk classification and therapeutic targets.Although ALL is the most common cancer in children,they are sensitive to standard chemotherapy and have a favorable prognosis that 5-year disease-free survival reached 90% in recent years.However,since the increasing occurrence of genetic abnormalities with poor prognosis and disappointed response to chemotherapy,adults with ALL have a low overall survival rate of 30%-40% and a high risk of complications and relapse.Current therapies that target specific genetic alterations remain insufficient,except for tyrosine kinase inhibitors(TKIs)such as imatinib in the treatment of BCR-ABL1 positive leukemia.Therefore,the genetic basis of leukemogenesis needs to be profoundly characterized,and the discovery of novel genetic abnormalities and molecular targets is of great interest.In previous work,we identified a somatic JAK1 mutation in 4 adults with B-ALL using whole exome sequencing,further validation in an additional 53 ALL cases showed 3 newly identified JAK1 heterozygous mutations(A639G,S646 P and P960S).To clarify the role of JAK1 mutations in ALL would be of great significance in illustrating the molecular mechanisms of ALL and discovering the novel therapeutic targets.The JAK genes encode non-receptor tyrosine kinases,including 4 members,namely,JAK1,JAK2,JAK3,and TYK2,which transduce the signals through JAK/STAT pathway to regulate the expression of target genes,involved in cell proliferation,differentiation,immune regulation and other biological processes.JAKs also play a great role in hematopoiesis,more than half of the patients withmyeloproliferative neoplasms(MPNs)were discovered with a recurrent somatic JAK2V617 F mutation,which has confirmed to be a key factor in the pathogenesis of MPNs.Ruxolitinib,as a JAK1/2 inhibitor,has been approved for the treatment of moderate and high risk myelofibrosis by FDA.Several other gain-of-function mutations at exon 12 of JAK2 have also been reported in JAK2 V617F-negative MPNs,indicating that dysregulation of JAK2 is an important mechanism and effective therapeutic target of MPNs.As a member of the family,the role of JAK1 cannot be ignored.In order to confirm the function of JAK1 mutations in ALL,we investigated the prevalence and clinical relevance of JAK1 mutations in ALL,and studied the oncogenic ability in vitro and in vivo.Firstly,since the identified JAK1 mutations are mainly located in exon 14 and 21,we designed specific primers and sequenced an additional 152 ALL cases.JAK1 Y652 H and N973 K mutations were identified in 2 samples.Together,we discovered five JAK1 mutations in 209 patients with ALL,of which two mutations were detected in T-ALL,and another in B-ALL.Among them,Y652 H and P960 S have been reported in COSMIC database,and N973 K has been recorded in dbSNP database,while S646 P and A639 G were newly discovered mutations in this study.We also found that these affected residues are highly conserved through alignment of amino acid sequences of JAK1.In summary,we revealed that JAK1 mutations occur at a frequency of approximately 2.4%(5/209)in ALL,particularly in T-ALL but rarely in B-ALL(10.5% versus 1.6%,P<0.05).We also observed that four of the five patients with mutations had an early or late relapse and resistance to chemotherapy,indicating that JAK1 mutations are likely to be associated with an elevated relapse and resistance risk.Secondly,we selected three mutations(A639G,S646 P and P960S)for further functional studies in vitro,JAK1 V658 F mutation was used as the positive control,due to its known gain of function.Lentiviral vector plasmids expressing wild type or mutants of JAK1 were transiently transduced to HEK293 V cells,and the phosphorylation of downstream signals was measured by western blot.Data showed that S646 P,P960S and V658 F mutants constitutively activated the phosphorylation of JAK1 and downstream signals such as STAT3 and ERK1/2.Stable Ba F3 and NALM-6cell lines transduced with wild type or mutants of JAK1 were established by lentiviralsystem.Cell proliferation and survival curves were determined by CCK-8,suggesting that only S646 P and V658 F mutants could induce the IL-3-independent growth of BaF3 cells,and resistence to the apoptosis caused by withdrawing IL-3.Cell cycle histograms of transduced cells were assayed by flow cytometry,indicating that S646 P mutant induced a higher percentage in S/G2 phase and promoted the cell proiferation.These data indicated that JAK1 S646 P mutation is gain-of-function similar to V658 F,as it conferred malignant transformation and enhanced activation of downstream signaling pathways.In addition,we also investigated the sensitivity of JAK1 mutations to ruxolitinib in vitro.The results showed that ruxolitinib attenuated the phosphorylation of JAK1,STAT3 and ERK1/2,and inhibited the proliferation of transduced BaF3 cells but had no effect on K562 with BCR-ABL1 fusion gene.Moreover,BaF3 cells transduced with S646 P and V658 F mutants exhibited more sensitivity to ruxolitinib than other groups.Finally,to explore the oncogenic potential of JAK1 S646 P mutation in vivo,BaF3 cells transduced with vector,WT or JAK1 S646 P mutant were injected intravenously into nude mice.The Ba F3/S646 P mice exhibited statistically significant elevated white blood cells(WBC)counts and GFP-positive cells infiltration in peripheral blood after30 days post-inoculation.At the time of death,the intravenous injection of BaF3/S646 P cells induced enlargement of liver and spleen and an elevated percentage of GFP-positive cells and blasts in bone marrow relative to vector and WT controls,suggesting that S646 mutation could induce malignant transformation and promote the infiltration and invasion of the malignant cells in vivo.Our work revealed that somatic JAK1 S646 P mutation is a gain-of-function mutation in this study,which was confirmed to be of oncogenic ability in vitro and in vivo,and sensitive to inhibitor,might become a new therapeutic target.We suggest that extending this gene in molecular testing for patients with ALL will be valuable for prognosis evaluation and targeted therapy,especially since some refractory and relapsed patients may benefit from JAK inhibitor therapy.