Cytokine Receptor-like Factor Research Articles

Cytokine Receptor-like Factor 2 (CRLF2) Overexpression in Myeloid Malignancies Secondary to Multiple Isodicentric Y Chromosomes: A Novel Cytogenetic Entity?

Background:Cytokine receptor-like factor 2 (CRLF2) overexpression is well-defined in B-cell and Ph-like ALL (~50%) with the CRLF2/IGH (76%) and P2RY8/CRLF2 (17%) rearrangements being the most common. CRLF2 is located on the pseudoautosomal region or PAR1 of both the short arm of X (Xp22.3) and Y (Yp11.3) chromosomes and its over-expression has not been previously described in myeloid neoplasms. Multiple isodicentric Y chromosomes, which have two centromeres and two copies of the short-arm of the Y chromosome, have the potential to produce amplification of CRLF2, leading to overexpression. We describe three cases of myeloid neoplasia, two with a diagnosis of myelodysplastic syndrome (MDS) and one with a diagnosis of acute myeloid leukemia (AML), in which cytogenetic studies demonstrated multiple copies of an isodicentric Y chromosome. CRLF2 overexpression was anticipated and confirmed via RNA sequencing in one patient. The clinical significance of CRLF2 overexpression is profound as it is known to activate the JAK-STAT pathway and is potentially targetable using tyrosine kinase inhibitors (TKIs).Case series:A similar genetic pattern was identified in three older males with a known diagnosis of myeloid malignancy, two of whom had MDS and one AML. The first patient is a 57-year-old man with a diagnosis of NPM-1/FLT3 ITD wt AML. Diagnostic cytogenetic abnormalities included 46-53, X, idic(Y)(q11.2)x1-8[cp12]/46,XY[8]. The second case is that of an 88-year-old man with monoclonal gammopathy of undetermined significance (MGUS), which progressed to multiple myeloma (MM) with trisomy 11. Two years after his diagnosis of MM, he was diagnosed with MDS with ringed sideroblasts (MDS-RS) with less than 5 per cent blasts, and the diagnostic karyotype result was 50-52,X,idic(Y)(q11.22)x3-4,+12. He had been treated with lenalidomide, dexamethasone and bortezomib. The third case is that of a 71-year-old man with transfusion-dependent anemia diagnosed as MDS-EB-1, with cytogenetic abnormalities at diagnosis being 50-54,XY,+idic(Y)(q11.2)x2-4,+8,+21[cp10]/46,XY[10]. Next generation sequencing identified pathogenic mutations in ASXL1 (c.1900_1922del; p.Glu635Argfs*15, 14%), IDH2 (c.419G>A; p.Arg140Gln, 37%) and SRSF2 (c.284_307del; p.Pro95_Arg102del, 36%). A bone marrow sample from this patient was subjected to RNA sequencing, and demonstrated overexpression of CRLF2, but not of SRY; confirming our hypothesis of multiple dicentric Y chromosomes resulting in CRLF2 overexpression (Figure 1).Discussion and Conclusions:Activation of JAK-STAT signaling is seen in Philadelphia chromosome positive (Ph) B- lymphoblastic lymphoma/leukemia (B-ALL) or Ph- like B- ALL and is therapeutically targetable with TKIs. Further, rearrangement of CRLF2 is not only associated with mutation of JAK kinases but also alteration of IKZF1, hispanic/latino ethnicity and a poor outcomes in pediatric B- ALL. Although the IGH-CRLF2 and P2RY8-CRLF2 rearrangements have been seen in up to 76 and 17 per cent of cases of adult Ph-like B-ALL cases respectively, there exist no reports of CRLF2 expression abnormalities in myeloid neoplasms (Jain N et al. Blood 2017).With the help of our series we document that: 1. Multiple isodicentric Y chromosomes are a unique cytogenetic abnormality that merits recognition and further clinical characterization in myeloid neoplasia. 2. The presence of up to eight isodicentric Y chromosomes, with up to 16 copies of the Y short arm, suggests the possibility of CRLF2 overexpression. 3. RNA sequencing can help confirm these results; thus providing relapsed/refractory patients with potential targeted salvage therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hematological characteristics, cytogenetic features, and post-induction measurable residual disease in thymic stromal lymphopoietin receptor (TSLPR) overexpressed B-cell acute lymphoblastic leukemia in an Indian cohort.

The overexpression of cytokine receptor-like factor-2 (CRLF2) identified by anti-thymic stromal lymphopoietin receptor/TSLPR flow cytometry (FCM) has been reported as a screening tool for the identification of BCR-ABL1-like B-cell acute lymphoblastic leukemia/B-ALL with CRLF2 re-arrangement. TSLPR expression was studied prospectively in consecutive 478 B-ALLs (≤ 12years(n = 244); 13-25years (n = 129); > 25years (n = 105)) and correlated with various hematological parameters and end-of-induction measurable residual disease (day 29; MRD ≥ 0.01% by 10-colorFCM). TSLPR positivity in ≥ 10% leukemic cells was detected in 14.6% (n = 70) of B-ALLs. CRLF2 re-arrangement was detected in eight cases (11.4%) including P2RY8-CRLF2 (n = 6), and IgH-CRLF2 (n = 2) with a median TSLPR positivity of 48.8% and 99% leukemic cells, respectively. Recurrent gene fusions/RGF (BCR-ABL1 (17.1%); ETV6-RUNX1 (4.2%), TCF3-PBX1 (1.4%)), other BCR-ABL1-like chimeric gene fusions/CGFs (PDGFRB-rearrangement (2.9%), IgH-EPOR (1.4%)), CRLF2 extra-copies/hyperdiploidy (17.1%), and IgH translocation without a known partner (10%) were also detected in TSLPR-positive patients. CD20 positivity (52.9% vs 38.5%; p = 0.02) as well as iAMP21 (4.3% vs 0.5%; p = 0.004) was significantly more frequent in TSLPR-positive cases. TSLPR-positive patients did not show a significantly higher MRD, compared to TSLPR-negative cases (37% vs 33%). Increasing the threshold cut-off (from ≥ 10 to > 50% or > 74%) increased the specificity to 88% and 100% respectively in identifying CRLF2 translocation. TSLPR expression is not exclusive for CRLF2 translocations and can be seen with various other RGFs, necessitating their testing before its application in diagnostic algorithms. In patients with high TSLPR positivity (> 50%), the testing may be restricted to CRLF2 aberrancies, while patients with 10-50% TSLPR positivity need to be tested for both CRLF2- and non-CRLF2 BCR-ABL1-like CGFs.

Three new cases of Crisponi /cold induced sweating syndrome (CS/CISS1) in Turkish families

Crisponi syndrome/Cold Induced Sweating Syndrome 1 (CS/CISS1) is a rare, autosomal recessive, multisystemic disease. Hyperthermia attacks, abnormal contractions in the muscles of the face and oropharynx, respiratory distress, camptodactyly, and swallowing difficulty are the main features of the condition in the neonatal period. Patients experience cold-induced sweating attacks and progressive kyphoscoliosis in childhood and adolescence. Mutations in the cytokine receptor like factor 1 (CRLF1) gene causes the CISS1 (Cold- induced sweating syndrome type 1) disease (over 95% of patients). CRLF1 is located in the ciliary neurotrophic factor receptor (CNTFR) pathway, which plays an important role in development and maintenance of neurons in the nervous system. In this study three patients from Turkey, clinically and molecularly diagnosed with CS/CISS1, are presented. Hyperthermia, swallowing difficulty, camptodactyly and pursing of the lips were present in all patients, and foot deformity in one patient. In the first patient a homozygous nonsense mutation NM_004750.5: c.531G > A; p.(Trp177Ter) in the 4th exon was detected. In the second patient a homozygous nonsense mutation NM_004750.5: c.776C > A; p.(Ser259Ter) in the 5th exon was detected. The third patient was homozygous for a missense mutation NM_004750.5: c.935G > T; p.(Arg312Leu) in the 6th exon. Early diagnosis is very important in this syndrome since most patients die in the neonatal period. Therefore, physicians should be suspicious for this disease in patients with dysmorphic features, hyperthermia attacks, camptodactyly, pursing of lips while crying, and swallowing difficulty.

CRLF2 and IKZF1 abnormalities in Mexican children with acute lymphoblastic leukemia and recurrent gene fusions: exploring surrogate markers of signaling pathways.

The gene fusions BCR‐ABL1, TCF3‐PBX1, and ETV6‐RUNX1 are recurrent in B‐cell acute lymphoblastic leukemia (B‐ALL) and are found with low frequency in coexistence with CRLF2 (cytokine receptor‐like factor 2) rearrangements and overexpression. There is limited information regarding the CRLF2 abnormalities and dominant‐negative IKZF1 isoforms associated with surrogate markers of Jak2, ABL, and Ras signaling pathways. To assess this, we evaluated 24 Mexican children with B‐ALL positive for recurrent gene fusions at diagnosis. We found CRLF2 rearrangements and/or overexpression, dominant‐negative IKZF1 isoforms, and surrogate phosphorylated markers of signaling pathways coexisting with recurrent gene fusions. All the BCR‐ABL1 patients expressed CRLF2 and were positive for pCrkl (ABL); most of them were also positive for pStat5 (Jak2/Stat5) and negative for pErk (Ras). TCF3‐PBX1 patients with CRLF2 abnormalities were positive for pStat5, most of them were also positive for pCrkl, and two patients were also positive for pErk. One patient with ETV6‐RUNX1 and intracellular CRLF2 protein expressed pCrkl. In some cases, the activated signaling pathways were reverted in vitro by specific inhibitors. We further analyzed a TCF3‐PBX1 patient at relapse, identifying a clone with the recurrent gene fusion, P2RY8‐CRLF2, rearrangement, and phosphorylation of the three surrogate markers that we studied. These results agree with the previous reports regarding resistance to treatment observed in patients with recurrent gene fusions and coexisting CRLF2 gene abnormalities. A marker phosphorylation signature was identified in BCR‐ABL1 and TCF3‐PBX1 patients. To obtain useful information for the assessment of treatment in B‐ALL patients with recurrent gene fusions, we suggest that they should be evaluated at diagnosis for CRLF2 gene abnormalities and dominant‐negative IKZF1 isoforms, in addition to the analyses of activation and inhibition of signaling pathways.

Locust Hemolymph Conveys Erythropoietin-Like Cytoprotection via Activation of the Cytokine Receptor CRLF3.

The cytokine receptor-like factor 3 (CRLF3) is an evolutionary conserved class 1 cytokine receptor present in all major eumetazoan groups. Endogenous CRLF3 ligands have not been identified and the physiological responses mediated by mammalian CRLF3 are poorly characterized. Insect CRLF3 is activated by erythropoietin (Epo) and several related molecules that protect mammalian neurons from stress-induced apoptosis. However, insects neither express Epo nor “classical” Epo receptor. Cell-protective effects of insect hemolymph have been described for several species. In this study, we explored the possibility that the endogenous CRLF3 ligand is contained in locust hemolymph. PCR analyses confirmed expression of crfl3-transcripts in neurons and hemocytes of Locusta migratoria and Tribolium castaneum. Survival of locust hemocytes in primary cultures was significantly increased by supplementation of culture medium with locust hemolymph serum. Locust primary neuron cultures were also protected by locust hemolymph, though preceding exposure to fetal bovine serum changed the hemolymph dose-dependency of neuroprotection. Direct comparison of 10% hemolymph serum with recombinant human Epo in its optimal neuroprotective concentration revealed equivalent anti-apoptotic effects on hypoxia-exposed locust neurons. The same concentration of locust hemolymph serum also protected hypoxia-exposed T. castaneum neurons. This indicates that the neuroprotective factor in locust hemolymph is sufficiently conserved in insects to allow activation of neuroprotective receptors in different species. Locust hemolymph-induced neuroprotection in both L. migratoria and T. castaneum was abolished after RNAi-mediated suppression of crlf3-expression. In summary, we report the presence of a conserved endogenous cytokine in locust hemolymph that activates CRLF3 and connected anti-apoptotic processes in hemocytes and neurons. Identification and characterization of the CRLF3 ligand will promote knowledge about cytokine evolution and may unravel cell-protective agents with potential clinical application.

Characterization of CRLF2 Expression in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with several underlying genetic ab-normalities. Several studies have tried to elucidate the prognostic significance of cytokine receptor-like factor 2 (CRLF2) overexpression in pediatric B-cell precursor (BCP)-ALL; however, it is still controversial. CRLF2 expression was assessed by flow cytometry in 87 newly diagnosed BCP-ALL pediatric patients, and 80 age and gender-matched control group. Janus Kinase2 (JAK2) (R683) mutation analysis was also performed in those identified to have CRLF2 overexpression with adequate DNA samples by direct sequencing. CRLF2 overexpression was identified in 26/87 (29.9%) of our patients with cutoff set at mean fluorescence intensity (MFI = 3.8) using the Receiver Operating Characteristic (ROC) curve. There were no significant differences in the clinical and laboratory features between patients with high and low-CRLF2 expression, apart from thrombocytopenia which showed statistically significant association with the low-expression group (p = 0.041). Sequence analysis of samples with high CRLF2 expression (n = 23) revealed that 2/23 (8.7%) cases harbored the mutation JAK2 (R683). CRLF2 levels did not have a significant impact on either overall survival (OS) or disease free survival (DFS) (p = 0.601; p = 0.212, respectively). CRLF2 overexpression was not an adverse parameter in pediatric BCP-ALL patients. However, patients with CRLF2 overexpression may harbor the JAK2 mutation presenting a group that can benefit from targeted therapy by kinase inhibitors. The usage of CRLF2 expression to monitor minimal residual disease of BCP-ALL would be an area of interest for further evaluation.

Prognostic significance of Cytokine Receptor-Like Factor 2 (CRLF2) gene expressions in pediatric acute lymphoblastic leukemia.

Background: The Cytokine Receptor-Like Factor 2 (CRLF2) genes play an important role in early Bcell development. Aberrations in CRLF2 activate the JAK-STAT signaling pathway that contributes to B-cell Acute Lymphoblastic Leukemia (B-ALL). The prognostic significance of CRLF2 overexpression in various B-ALL risk subgroups has not been well established. Methods: Thirty patients with newly diagnosed childhood B-ALL were enrolled from the Egyptian population. All the patients in this study were assessed by detailed history taking, a complete clinical examination required radiological and routine laboratory investigations, and flow cytometry to detect the subtype of ALL. Peripheral Blood and bone marrow samples were taken from patients, and the CRLF2 expression was measured using RT-PCR. Results: The prevalence of CRLF2 overexpression status was analyzed, and the prognostic impact of CRLF2 overexpression on B-ALL was evaluated by assessing their influence on overall survival and event-free survival. CRLF2 overexpression was found in 56% of the whole cohort. The results showed that CRLF2 overexpression was associated with a poor outcome in unselected B-ALL. Conclusion: From the present study's findings, we can conclude that CRLF2 gene expression at diagnosis has a significant prognostic impact on the clinical outcome in childhood ALL.

Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. IMPLICATION: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.

High CRLF2 Expression Could Identify Acute Lymphoblastic Leukemia Patients with Poor Outcome but Not IKZF1

Background: Overexpression of cytokine receptor-like factor 2 (CRLF2) caused by different genetic aberrations has been observed in acute lymphoblastic leukemia (ALL) and correlated with poor outcome. Most patients with high CRLF2 expression are clustered in the Philadelphia-like (Ph-like) ALL subgroup. Ph-like ALL is reported to be associated with alterations in IKZF1 gene, encoding the transcription factor Ikaros. Aim: To identify CRLF2 and IKZF1 alterations in Egyptian patients with ALL and to determine their prognostic significance. Methods: Peripheral blood samples from 34 newly diagnosed ALL patients treated at an Egyptian tertiary oncology center and 14 controls were assessed for CRLF2 and IKZF1 mRNA expression using real-time polymerase chain reaction. Results: CRLF2 was significantly overexpressed in ALL patients compared to controls (p = 0.038). The response to treatment was significantly better in patients with low CRLF2 expression (p = 0.029). The rate of remission, relapse and induction death was 82%, 12% and 6% in the low CRLF2 expression group and 41%, 18% and 41% in the high expression one. Overall survival was significantly shorter among ALL with high CRLF2 (p = 0.034). IKZF1 expression level did not differ significantly between patients and controls. Patients with low IKZF1 exhibited significantly higher leucocytic count and lower platelet count (p = 0.038 and 0.044, respectively). IKZF1 overexpression did not correlate significantly with response to treatment or survival. Conclusion: High CRLF2 expression was associated with poor outcome among ALL patients. Further research is needed to improve the diagnostic and therapeutic approaches in ALL patients with poor prognosis.

A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.

Inducible Knockout of HMGN1 in an In Vivo xenograft Model Reduces Down Syndrome Leukemic Burden and Increases Survival Outcomes

Introduction Down Syndrome (DS) patients are at high risk of developing hematological malignancies and ~10% are born with a pre-leukemic disorder characterised by the overproduction of immature megakaryoblasts. Children with DS have a 20-fold increased risk of developing acute lymphoblastic leukemia (ALL) of which 60% are associated with high expression of cytokine receptor like factor 2 (CRLF2) and of these, ~9% acquire the aggressive CRLF2 p.F232C mutation. DS-ALL children also experience high treatment toxicity and high relapse rates compared to non-DS leukemia patients. Genes on chromosome 21 including the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) are likely to play a role in DS leukemogenesis and may be targets for a personalized treatment approach. We aimed to determine if HMGN1 is necessary for leukemic cell proliferation using an inducible CRISPR/Cas9 guide (g)RNA murine xenograft model. Methods A DS leukemic cell line model was created using the human trisomy 21 megakaryoblastic SET-2 cell line harboring JAK2 p.V617F; the only trisomy 21 leukemic cell line currently available. SET-2 cells were transduced with CRLF2 p.F232C to model an aggressive DS-ALL mutation. NOD.Cg-Prkdcscid,Il2rgtm1Wjl/Szj (NSG) mice were each injected with 3x105 SET-2 CRLF2 p.F232C CRISPR/Cas9 cells expressing luciferase in 3 groups; Cas9 only control, HMGN1 gRNA, or JAK2 positive control gRNA. Doxycycline was administered post leukemic engraftment to induce the gRNAs and create a knockout (KO) and leukemic burden was monitored by bioluminescent imaging (BLI) twice weekly for the remainder of the experiment. Once Cas9 control mice became moribund, they were culled along with 50% of the JAK2 or HMGN1 KO mice and complete blood counts were performed. Bone marrow (BM), spleen and liver sections were stained with hematoxylin and eosin (H&E) and survival analysis was carried out for remaining JAK2 or HMGN1 KO mice. RQ-PCR was used to detect HMGN1 expression levels in KO mice organs at endpoint and DNA was extracted from cells harvested from each organ to undertake a gene editing analysis. Results Leukemic engraftment in mouse BM was observed 10 days post transplant with a radiance signal of ~1 x 104p/s/cm2/sr, therefore gRNAs were induced on day 11. On day 20, a significant reduction in tumor burden was detected in JAK2 and HMGN1 KO mice compared to Cas9 control mice (Fig. 1, Cas9: 8.4x105±1.7x105; JAK2 KO: 2.7x104±8.9x103; HMGN1 KO: 1.5x105±1.7x104 p/s/cm2/sr, prone: p<0.001, supine: p=0.005). Blood counts at day 35 indicated similar white cell counts across Cas9, JAK2 and HMGN1 KO mice, however, the Cas9 mice demonstrated thrombocytopenia and anemia (platelet count: 705±43 K/µL, HCT: 22.5±2%) which was rescued in JAK2 and HMGN1 KO mice (JAK2 KO platelet count: 3046±775 K/µL, p<0.001; HCT: 47±6.8%, p=0.002; HMGN1 KO platelet count: 1503±83 K/µL, p<0.001; HCT: 38±3.4%, p=0.004). JAK2 and HMGN1 KO mice had reduced spleen weight (JAK2 KO: 46±2 mg, p=0.019; HMGN1 KO: 51±6 mg, p=0.046; Cas9: 81±7 mg) and liver weight compared to Cas9 control mice. Megakaryoblast infiltration identified with H&E staining was evident in the BM, spleen and liver of Cas9 control mice, whereas megakaryoblasts were not observed in JAK2 or HMGN1 KO mice organs. Similarly, RQ-PCR demonstrated a 24% decrease in HMGN1 expression in the BM, 99% in the spleen and 92% in the liver; and a 38% decrease in JAK2 expression in the BM, 99% in the spleen and 70% in the liver of HMGN1 and JAK2 KO mice respectively compared to Cas9 control mice. Significantly, survival analysis of the remaining JAK2 and HMGN1 KO mice indicated a substantial survival advantage from 35 days (Cas9) to 62 and 56 days respectively for JAK2 and HMGN1 KO mice (p=0.0009). Conclusion Our CRISPR/Cas9 DS leukemic xenograft HMGN1 KO model demonstrates the important role of HMGN1 in CRLF2 p.F232C DS leukemia. Significantly, HMGN1KO decreased the leukemic burden of mice to the same extent as the JAK2 (SET-2 driver gene) KO. The HMGN1 KO mitigated ALL phenotypes including hepatosplenomegaly, anemia and thrombocytopenia, preventing leukemic progression and resulting in a significant survival advantage over Cas9 control mice. As HMGN1 has a distinct role in proliferation and survival of DS leukemic cells, it is a potential candidate for targeting with a pharmacological inhibitor as a personalized treatment for DS leukemia patients. Disclosures White: Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria.

Outcomes of Patients with CRLF2-Overexpressing Acute Lymphoblastic Leukemia without Down Syndrome: A Report from the Children's Oncology Group

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) occurs in 5-30% of children and adolescents/young adults (AYAs) with B-ALL, is driven by genetic alterations that induce constitutive cytokine receptor and kinase signaling, and is associated with poor clinical outcomes across the older pediatric-to-adult age spectrum (Tasian Blood 2017c, Reshmi Blood 2017, Roberts Blood 2018). Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of Ph-like ALL cases with frequently co-occurring JAK2 or JAK1 point mutations or IL7R indel mutations. This study reports the clinical outcomes of children and AYAs with newly-diagnosed National Cancer Institute (NCI) standard-risk (SR) or high-risk (HR) CRLF2+ ALL without Down syndrome treated on four successive Children's Oncology Group (COG) phase 3 clinical trials from 2003 to 2018. Methods: We retrospectively assessed demographic characteristics, laboratory data, and clinical outcomes of 3757 patients with B-ALL treated on COG trials AALL0331 and AALL0932 (SR) and AALL0232 and AALL1131 (HR) whose diagnostic leukemia specimens were analysed by low-density microarray (LDA), fluorescence in situ hybridization, polymerase chain reaction (PCR), and/or anchored multiplex PCR testing (Harvey and Tasian Blood Advances 2020). Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at the end of consolidation for a subset of EOI MRD+ patients. Results: We identified 77/1541 (5.0%) SR and 244/2216 (11.0%) HR patients with CRLF2+ B-ALL in this cohort. Amongst those with diagnostic leukemia specimens analysed by LDA, 57/72 (79.2%) of SR CRLF2+ and 175/213 (82.2%) of HR CRLF2+ patients were positive for the Ph-like gene expression profile with an 8-gene score ≥0.5. P2RY8-CRLF2 fusions and IGH-CRLF2 translocations were detected in 64/77 (83.1%) and 10/77 (13.0%) of SR CRLF2+ patients and in 98/244 (40.2%) and 103/244 (42.2%) of HR CRLF2+ patients, respectively. CRLF2 rearrangements or F232C mutations were not found in the remaining 3 SR and 43 HR CRLF2+ patients, although other Ph-like alterations were discovered in some (n=3 IGH-EPOR fusions, 1 IL7R indel). Importantly, CRLF2+ vs non-CRLF2-overexpressing (CRLF2-) status was associated with older age (10.8 ±6.5 vs 7.8 ±5.8 years [mean ±SD], p<0.0001), leukocytosis (diagnostic white blood cell count 77.5 ±98.5 vs 49.8 ±119.4 x 10e9/L [mean ±SD], p<0.0001), and higher rates of EOI MRD positivity at a ≥0.01% threshold (47.9% vs 30.1%, p<0.0001), which appeared largely driven by the Ph-like HR cohort as expected (57.9% MRD+ vs 42.1% MRD- in HR CRLF2+ and 44.6% MRD+ vs 55.4% MRD- in SR CRLF2+ patients, p<0.003). Overall, CRLF2+ patients had inferior 5-year event-free survival (EFS; 63.3% ±3.1 vs 82.1% ±0.7, p<0.0001) and overall survival (OS; 79.6% ±2.6 vs 90.5% ±0.6, p<0.0001) compared to CRLF2- patients (Figure 1A-B) and a greater 5-year cumulative incidence of relapse (CIR; 30.4% ±2.7 vs 13.2% ±0.6, p<0.001). While 5-year EFS and OS were particularly poor in Ph-like CRLF2+ HR patients (56.3% ±4.6 and 75.4% ± 3.9, respectively) and non-Ph-like CRLF2+ HR patients (EFS 63.7% ±10.2 and OS 74.4% ±8.9), outcomes for Ph-like CRLF2+ SR (EFS 87.2% ±4.5 and OS 94.5% ±3.1) and non-Ph-like CRLF2+ SR patients (EFS 86.2% ±9.3 and OS 100%) were quite good (p<0.0001 for both EFS and OS; Figure 1C-D). Discussion: Patients with newly-diagnosed CRLF2+ B-ALL treated on frontline COG trials have higher rates of EOI MRD positivity, inferior survival, and increased CIR compared to their CRLF2- counterparts. EFS is especially poor in children and AYAs with NCI HR CRLF2+ ALL, particularly those with the Ph-like expression profile. Conversely, outcomes for children with NCI SR CRLF2+ ALL are reasonably favourable, irrespective of Ph-like status. Development of successful treatment strategies to decrease relapse and to improve survival remains a major therapeutic gap for NCI HR CRLF2+ ALL patients. Current clinical trials are studying the potential efficacy of kinase inhibitor addition to chemotherapy for children, adolescents, and adults with HR Ph-like ALL harboring CRLF2 rearrangements/other JAK pathway alterations or ABL class kinase fusions (NCT0240717, NCT02723994, NCT02883049, NCT03571321). Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria. Raetz:Pfizer: Research Funding; Celgene/BMS: Other. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Outcomes of Patients with Down Syndrome and CRLF2-Overexpressing Acute Lymphoblastic Leukemia (ALL): A Report from the Children's Oncology Group (COG)

Background: Patients with Down syndrome (DS) have an approximately 10-fold increased risk of developing ALL, and the spectrum of genetic alterations differs from that of non-DS ALL. Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of DS ALL, compared to only 5-10% CRLF2+ cases in non-DS children and adolescents. JAK2 or JAK1 mutations co-occur in about half of CRLF2+ cases in both DS and non-DS ALL. The prognostic significance of CRLF2+ ALL also appears to differ in the limited data reported to date, with less adverse impact in patients with DS compared to non-DS ALL. Here, we report the clinical characteristics and prognostic significance of B-ALL with CRLF2 overexpression and JAK alterations in children and adolescents/young adults (AYA) with DS who were treated on Children's Oncology Group (COG) clinical trials from 2003-2016. Methods: We analyzed clinical, laboratory, and outcome data for 317 patients with DS B-ALL treated on standard risk (SR) trials AALL0331 and AALL0932 and high risk (HR) trials AALL0232 and AALL1131, for whom CRLF2 status and rearrangement partners (IGH or P2RY8) were ascertained by flow cytometric assessment of surface expression; fluorescence in situ hybridization; and/or polymerase chain reaction (PCR) testing. JAK mutations were ascertained in a subset by PCR and sequencing. Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at end of consolidation (EOC) for a subset of EOI MRD+ patients. Results: We identified 168/317 (53.0%) CRLF2+ cases, and among those assessed for CRLF2 partner, 17/73 (23.3%) were IGH-CRLF2 and 56/73 (76.7%) were P2RY8-CRLF2. In the subset of 165 cases tested for JAK mutations (85 CRLF2- and 80 CRLF2+), 42/165 (25.4%) had JAK mutations, all of which co-occurred in CRLF2+ cases. CRLF2 positivity was significantly associated with younger age at diagnosis: 140/168 (83.3%) of CRLF2+ cases were under 10 years old, versus 106/149 (71.1%) of CRLF2- cases, p<0.01. P2RY8-CRLF2 cases were significantly more likely than IGH-CRLF2 cases to be associated with age under 10 years at diagnosis (92.9% vs 29.4%, p<0.0001), initial white blood count (WBC) <50x103 (80.4% vs 52.9%, p<0.024), National Cancer Institute (NCI) standard risk status (73.2% vs 23.5%, p<0.0002), and day 29 MRD <0.01% (67.9% vs 37.5%, p<0.028). There were no significant associations between JAK mutation status and any clinical features assessed (sex, initial WBC, central nervous system or testicular involvement, NCI risk group, or EOI/EOC MRD). Among patients with neutral cytogenetics (defined as neither favorable [ETV6-RUNX1, double trisomies of chromosomes 4 and 10] nor unfavorable [BCR-ABL1, KMT2A-rearranged, hypodiploid, iAMP21]), survival did not significantly differ between CRLF2+ and CRLF2- patients (Figure 1A,B), either for 5-year event-free survival (EFS) (79.1 +3.6% vs 77.6 +4.7%, p=0.856) or 5-year overall survival (OS) (89.5 ±2.8% vs 84.9 ±4.1%, p=0.674). There were also no significant differences in EFS or OS based on JAK mutation status or CRLF2 partner, although there were trends toward poorer outcomes in the CRLF2+/JAK+ and IGH-CRLF2 subgroups (Figure 1C). Finally, there was no significant difference in 5-year cumulative incidence of relapse (CIR) according to CRLF2 status (CRLF2+ 16.0 +3.0%, CRLF2- 10.6 +2.6%, p=0.179), although there were non-significant trends toward more relapses in CRLF2+ cases overall and in the NCI HR subgroup analysis. Discussion: Whereas CRLF2 and JAK alterations are associated with higher MRD, poorer survival, and increased CIR in patients with high-risk ALL without DS, these alterations do not demonstrate strong adverse prognostic impact in children and AYAs with DS-ALL treated on recent frontline COG trials, although larger sample sizes are needed to adequately assess for possible poorer prognoses associated with the CRLF2+/JAK+ and IGH-CRLF2 subgroups. Regardless, given the frequency of these targetable lesions and the increased risk of relapse and chemotherapy-associated toxicities in patients with DS-ALL, targeted therapies currently under investigation for these genetic lesions may be beneficial to replace some intensive blocks of therapy in DS-ALL with CRLF2 and/or JAK alterations, both to enhance anti-leukemic efficacy and decrease intensive chemotherapy-associated toxicities. Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau. Raetz:Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria.

Crisponi/Cold Induced Sweating Syndrome Type 1 With a Private Cytokine Receptor Like Factor 1 (CRLF1) Mutation in an Indian Family

Crisponi/Cold Induced Sweating Syndrome Type 1 With a Private Cytokine Receptor Like Factor 1 (CRLF1) Mutation in an Indian Family

ALL-228: CRLF2 and IKZF1 Expression in Egyptian Acute Lymphoblastic Leukemia Patients

Objective Overexpression of cytokine receptor-like factor 2 (CRLF2) caused by different genetic aberrations has been detected in ALL and is linked to poor outcome. The majority of cases having high CRLF2 expression are clustered in the Philadelphia-like (Ph-like) ALL subgroup. Furthermore, Ph-like ALL is accompanied by alterations of the IKZF1 gene, encoding the transcription factor Ikaros. Thus, it was important to recognize and identify these cases at presentation. Materials and Methods We investigated clinical characteristics and the prognostic significance of mRNA expression levels of CRLF2 and IKZF1 alterations in a group of Egyptian patients with ALL. Peripheral blood samples from 34 newly diagnosed ALL cases, together with 14 healthy controls, were assessed for CRLF2 and IKZF1 expression by qRT-PCR. Results CRLF2 mRNA level showed significant overexpression in ALL patients in comparison to healthy controls (P = 0.038). Overall survival was shorter among B-ALL patients, with high CRLF2 (6.1 months versus 19.4 months, respectively, P = 0.04). Moreover, patients with low IKZF1 exhibited significantly higher total leucocytic counts and lower platelet counts (P 0.038 and 0.044, respectively). Conclusion High CRLF2 expression was linked to poor survival among B-ALL cases. Identification of Ph-like ALL should be recognized as a distinct, high-risk entity, and future research on improving diagnostic and therapeutic approaches is required in our locality.

Abstract 3234: Multi-antigen targeting of CD19, CD22 and TSLPR to prevent Ph-like ALL resistance

Abstract Background: Despite high remission induction rates after CD19 chimeric antigen receptor T cell (CART) therapy in patients with B-acute lymphoblastic leukemia (ALL), many patients eventually relapse with loss of CD19 antigen. Strategies to overcome antigen-loss relapse include co-targeting of multiple antigens and selection of alternative functionally important surface targets. The thymic stromal lymphopoietin receptor (TSLPR), encoded by CRLF2 (cytokine receptor-like factor 2), is overexpressed in >50% of patients with Ph-like ALL and is associated with constitutively active JAK/STAT signaling targetable by ruxolitinib. Our groups previously demonstrated potent activity of TSLPRCART against TSLPR+ ALL (Qin Blood 2015). Our current study aims to develop (1) multi-specific CAR T cells targeting TSLPR and CD19 or CD22 and (2) TSLPRCART and ruxolitinib combinatorial approaches to decrease antigen-negative relapse potential after CART therapy. Methods: Lentiviral transduction of activated healthy donor T cells was used to introduce monovalent TSLPR or CD19 CARs and bivalent CD19xTSLPR or CD22xTSLPR CARs. CAR expression was quantified via flow cytometry. CRISPR/Cas9-modified Nalm-6 (human B-ALL) cells were transduced with a TSLPR expression vector to generate cell lines expressing combinations of CD19, CD22, and TSLPR antigens. In vitro functionality was assessed using enzyme linked immunosorbent assays to detect IL-2 and IFN-γ production following CART co-culture with Nalm-6. In vivo evaluation was performed against luciferase-transduced Nalm-6 murine xenograft models treated with 5e6 CART and monitored by bioluminescent imaging. TSLPRCART was also tested against MUTZ5 (TSLPR-overexpressing Ph-like ALL) cells in vitro and in vivo in combination with ruxolitinib to determine effects on CAR T cells. Results: TSLPRCART and CD19xTSLPRCART induced in vitro cytokine production when co-cultured with CD19+/TSLPR+ Nalm-6 cells with higher levels than those produced by CD19CART. Greater cytokine production was observed with co-incubation of TSLPR+ Nalm-6 cells with CD22xTSLPRCART vs TSLPRCART. In vivo studies demonstrated equivalent leukemia eradication by TSLPRCART and CD19CART compared to CD19xTSLPRCART in CD19+/TSLPR+ Nalm-6 xenograft models. Additional in vivo studies in MUTZ5 xenograft models co-treated with TSLPRCART and ruxolitinib demonstrated impaired T cell expansion and cytokine production, which could be overcome via higher CART cell dose or delayed ruxolitinib administration. Conclusions: TSLPRCART is a robust immunotherapeutic strategy for CRLF2-rearranged Ph-like ALL. We demonstrate potent in vitro and in vivo functionality of novel bivalent CD19xTSLPRCART and CD22xTSLPRCART. Ongoing studies are also evaluating bicistronic CD19xTSLPR CAR constructs. We further demonstrate that combining TSLPRCART with ruxolitinib moderately blunts T cell expansion and cytokine production, which could be an attractive strategy for management of CAR T cell-associated toxicities such as cytokine release syndrome. Citation Format: Savannah Ross, Asen Bagashev, Dina Schneider, Peirong Hu, Sarah Tasian, Terry Fry. Multi-antigen targeting of CD19, CD22 and TSLPR to prevent Ph-like ALL resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3234.

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL.

Children and adults with Philadelphia chromosome-like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor-like factor 2-rearranged (CRLF2-rearranged) Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe an adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK, and PI3K signaling consistent with activated B cell receptor (BCR) signaling, although they do not express cell surface μ-heavy chain (μHC). Combinatorial targeting of JAK/STAT, PI3K, and "BCR-like" signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.

Generation of induced pluripotent stem cell lines from a Crisponi/Cold induced sweating syndrome type 1 individual.

Cytokine receptor like factor 1 (CRLF1) is the gene implicated, when mutated, in Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1). Here, we report the establishment of induced pluripotent stem cell lines (iPSCs) from fibroblasts of a Turkish CS/CISS1 individual with a homozygous variant in CRLF1 (c.708_709delinsT; p.[Pro238Argfs*6]). This variant is the most frequent variant associated to CS/CISS1 in the Turkish population. These patient derived iPSC lines show all pluripotency markers, a normal karyotype and the ability to differentiate into the three germ layers.

CRLF2 (Cytokine receptor like factor 2)

CRLF2 (Cytokine receptor like factor 2)

Oncogenesis of CRLF2 Overexpression and Effect of JAK2 Inhibitor in CRLF2 Overexpressed B-Cell Acute Lymphoblastic Leukemia

Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P<0.0001). Patients with CRLF2 overexpression had a significantly higher WBC count (53*10^9/L vs. 29.5*10^9/L, P=0.041). Survival analysis showed that the patients with CRLF2 overexpression had a worse EFS and OS, the differences were statistically significant (11 months vs. 26 months, P=0.043 and 15 months vs. 32 months, P=0.015). Also, the CRLF2 expression is determined with flow cytometry after staining with FITC-CRLF2 antibody in 28 samples. The correlation analysis was performed on the CRLF2 expression detected by qPCR and flow cytometry, respectively. A significant positive correlation of the two methods was observed(r=0.957, P<0.0001). These data not only indicate that CRLF2 overexpression is a marker of poor outcome, but also reveal the qPCR might be a simple and quick method for screening CRLF2 overexpression in the clinic compared to flow cytometry which is commonly used. We further found that expression of CRLF2 or CRLF2 F232C mutant into Nalm6 and 697 B-ALL cells dramatically increase the CRLF2 mRNA level, which is 69 times than vector-only control. Moreover, CRLF2 or CRLF2 F232C significantly promotes the cell proliferation of Nalm6 and 697 cells compared to vector only (P<0.001). In addition, JAK2 inhibitor (BBT594) treatment showed the significant dose-dependent cell proliferation arrest and clonogenic inhibition in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells compared to vector-only control. Furthermore, in vivo we observed the 5-fold higher signal intensity of leukemia engraftment in the mice injected with Nalm6-CRLF2-luc or Nalm6-F232C-luc compared to that of Nalm6-vector-luc control 1-3 weeks after the injection(P<0.001). The Nalm6-CRLF2-luc and Nalm6-F232C-luc infiltrations were observed in bone marrow, central nervous system, liver and spleen of the mice. Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.