Cytokine Receptor-like Factor Research Articles

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia

This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p=0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

High occurrence of CRLF2 abnormalities in Mexican children with B-cell acute lymphoblastic leukemia.

The P2RY8-CRLF2 and IGH-CRLF2 rearrangements induce the overexpression of cytokine receptor-like factor 2 (CRLF2) and have been associated with relapse and poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Additionally, they are frequently documented in high-risk Hispanic populations. To better understand the potential causes of the adverse prognosis of childhood B-ALL in Mexico, we analyzed these rearrangements and the CRLF2 mRNA and protein levels in 133 Mexican children with B-ALL. We collected bone marrow samples at diagnosis and evaluated the CRLF2 gene expression by qRT-PCR and the total CRLF2 protein by flow cytometry. P2RY8-CRLF2 and IGH-CRLF2 were detected by RT-PCR and FISH, respectively. The median time of follow-up to determine the prognostic significance of the CRLF2 abnormalities was three years. In 82% of the participants, the mRNA levels correlated with the cell-surface and intracellular CRLF2 protein levels. The P2RY8-CRLF2 rearrangement was present in 31.5% (42/133) of the patients, while the IGH-CRLF2 rearrangement was detected in 13.5% (9/67) of patients with high expression of CRLF2 (6.8% of the total sample). CRLF2 copy number variations (gain) were also detected in 7.5% (5/67) of patients with high protein levels. The overall survival (OS) presented significantly lower rates in patients with high white blood cell count (≥50x109/L) regardless of CRLF2 expression, but high levels of CRLF2 gene expression appears to contribute to the reduction of OS within this group of patients. In conclusion, in our cohort, a high occurrence of CRLF2 abnormalities was documented, particularly the P2RY8-CRLF2 rearrangement, which might represent a characteristic of the Mexican population. Targeted therapy to treat this group of patients could improve OS.

P1421: BIMODAL TARGETING OF CYTOKINE RECEPTOR-LIKE FACTOR 2 (CRLF2) WITH JAK INHIBITION AND CHIMERIC ANTIGEN RECEPTOR T CELL IMMUNOTHERAPY IN DOWN SYNDROME ACUTE LYMPHOBLASTIC LEUKEMIA

Background:CRLF2 (cytokine receptor-like factor 2; encoding the thymic stromal lymphopoietin receptor [TSLPR]) rearrangements occur in 50% of children with Down Syndrome-associated B-acute lymphoblastic leukemia (DS-ALL). These genetic alterations induce constitutive JAK/STAT and other kinase signaling that may be targetable by the JAK1/2 inhibitor ruxolitinib. Patients with DS-ALL experience substantial toxicity with conventional chemotherapy and particularly with hematopoietic stem cell transplantation (HSCT). The Children’s Oncology Group AALL1521 clinical trial (NCT02723994) is investigating the potential superior efficacy of ruxolitinib addition to chemotherapy for pediatric patients with CRLF2-rearranged/JAK pathway-mutant BCR-ABL1-like ALL, but children with DS-ALL are not eligible to participate in this study Successful development of precision medicine approaches that can increase cure, reduce treatment toxicity, and avoid HSCT remain a priority for children with D-ALL. Aims: We hypothesized that dual CRLF2 targeting with ruxolitinib and cellular immunotherapy could improve anti-leukemia activity against CRLF2-rearranged (R) DS-ALL. Methods: We previously reported preclinical activity of TSLPR-redirected chimeric antigen T cells with 4-1BB/CD3z costimulatory domains [TSLPRCART] against CRLF2-overexpressing ALL cells (Qin Blood 2015). In the present studies, we investigated the effects of TSLPRCART +/- ruxolitinib co-treatment in vitro and in vivo against CRLF2-R human ALL cell lines and childhood DS-ALL patient-derived xenograft (PDX) models (n=4). Results:In vitro incubation of CD28/CD3 bead-activated normal human T cells or TSLPRCART with ruxolitinib significantly diminished IL-2 and IFN-ɣ cytokine production, although did not induce overt apoptosis of normal or engineered T cells. As expected, treatment of CRLF2-R DS-ALL PDX models with TSLPRCART potently inhibited in vivo leukemia proliferation and induced long-term ‘cure’ of animals. However, simultaneous co-treatment of engrafted mice with ruxolitinib and TSLPRCART led to markedly diminished in vivo T cell proliferation and persistence, blunted cytokine production, and facilitated eventual leukemia relapse (representative data in Figure 1). These deleterious effects could be abrogated by 14-day delayed sequencing of ruxolitinib after TSLPRCART activation and expansion and were further fully reversible after ruxolitinib withdrawal, resulting in renewed TSLPCART functionality over time and upon leukemia rechallenge. Image:Summary/Conclusion: TSLPRCART is a promising cellular immunotherapeutic approach for DS-ALL and for BCR-ABL1-like ALL given shared features of CRLF2 rearrangement and cell surface protein overexpression. In the present studies, we demonstrate that ruxolitinib co-administration in close proximity impaired in vivo TSLPRCART-induced DS-ALL cell killing but was also beneficial in protection against life-threatening hyperinflammation/cytokine release syndrome (CRS) in dual-treated animals. These results suggest potential for both anti-ALL efficacy and CRS dampening when JAK inhibition and TSLPRCART are properly time-sequenced and may have broader applicability for toxicity mitigation with other cellular immunotherapies. Studies of CAR T cell immunotherapy and other kinase inhibitors in additional CRLF2-rearranged and non-rearranged DS-ALL models are ongoing.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.

Abstract 1528: Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of leukemia in children. B-ALL characterized by cytokine receptor-like factor 2 (CRLF2) overexpression (CRLF2 B-ALL) has a survival rate of &amp;lt;30% and is the highest risk sub-group of B-ALL in both adults and children. CRLF2 is a receptor component for the cytokine thymic stromal lymphopoietin (TSLP). TSLP plays a role in the survival and proliferation of B-cell precursors, thus explaining the oncogenic role of increased CRLF2 signaling in CRLF2 B-ALL. To our surprise, we found that high-levels of TSLP eliminated leukemia cells in patient-derived xenograft (PDX) models of CRLF2 B-ALL. CRLF2 and IL-7 receptor-alpha (IL-7Ra) form the heterodimer type-I cytokine receptor for TSLP cytokine. Binding of TSLP to its CRLF2 receptor complex induces JAK-STAT5 and PI3K-AKT pathway signals. TSLP shares the IL-7Ra with Interleukin 7 (IL-7) which has a heterodimer receptor consisting of IL-7Ra and the common gamma chain. High-levels of IL-7 (50 ng/ml) have been shown to induce IL-7Ra internalization and degradation in T-cells. We hypothesize that high-level TSLP induces internalization and degradation of IL-7Ra leading to CRLF2 signal inhibition, death of CRLF2 B-ALL cells and the anti-leukemia effects that we have observed in PDX mice. To test this hypothesis, we treated CRLF2 B-ALL cell lines with different TSLP concentrations to observe the effect of TSLP on its receptor and CRLF2 signaling. Flow cytometry data showed that continuous or a pulse of high-dose TSLP induced a loss of surface IL-7Ra expression for up to 24 hours. Phosphorylation assays showed that cells cultured with high-dose TSLP were unresponsive to subsequent TSLP-induced phosphorylation events (pSTAT5 and pRPS6), indicating CRLF2 signal inhibition. In conclusion, high-dose TSLP induces loss of (IL-7Ra) and inhibition of CRLF2 signaling. These results suggest that TSLP exerts its anti-leukemia effects by shutting down CRLF2-mediated signals possibly via the loss of the IL-7Ra receptor component. Citation Format: Hossam Alkashgari, Cornelia Stoian, Caleb Ruiz-Jimenez, Jacqueline Coats, Carlos A. Casiano, Sinisa Dovat, Kimberly J. Payne. Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1528.

Evaluating the Role of Cytokine Receptor-like Factor 2 and Janus Kinase 2 in Adult Acute Lymphoblastic Leukemia

AIM: The aim of the present study was to assess the diagnostic, prognostic, and predictive roles of the cytokine receptor-like factor 2 (CRLF2) and the Janus Kinase 2 (JAK2) genes expression in adult acute lymphoblastic leukemia (ALL) patients. METHODS: The expression levels of CRLF2 and JAK2 genes were evaluated in the bone marrow (BM) samples of 105 adult ALL patients, compared to 12 healthy controls. The data were correlated to the patients’ relevant clinic-pathological features, response to treatment and survival rates. RESULTS: There was a significant overexpression of JAK2 in ALL patients compared to the control group [0.04 (0–160.8) and 0.006 (0–0.009), respectively, p &lt; 0.001]. Similarly, CRLF2 was overexpressed in ALL patients in comparison to control subjects [0.008 (0–78.2) and 0.0005 (0–0.006), respectively, p &lt; 0.001]. The sensitivity, specificity, and the area under curve (AUC) for JAK2 were 78.1%, 81.8%, and 0.796, respectively (p &lt; 0.001), and that of CRLF2 were 92.4%, 90.9%, 0.958, respectively (p &lt; 0.001). When combining both JAK2 and CRLF2 for the diagnosis of ALL patients, it revealed 90.9% sensitivity, 91.4% specificity, and AUC of 0.957 (p &lt; 0.001). The JAK2, CRLF2, or their combined expression associated significantly with the increased expression of MHC-II (p = 0.015, 0.001, and 0.004, respectively). However, they had no significant impact on patients’ response to treatment, overall (OS), and disease-free survival (DFS) rates (p &gt; 0.05 for all). CONCLUSION: JAK2 and CRLF2 could be a potential useful diagnostic molecular marker for ALL patients, which allow them to be successful targets for ALL therapy.

Thymic stromal lymphopoietin (TSLP) is a potent pro-inflammatory mediator which is epigenetically deregulated in endometriosis

ObjectiveEndometriosis is an estrogen-dependent disease characterized by the presence of endometriotic tissue outside the uterine cavity, the condition that immunological factors play important roles in its pathogenesis. Thymic stromal lymphopoietin (TSLP) is an interleukin 7–like cytokine that triggers dendritic cell–mediated T helper2 inflammatory responses. TSLP receptor, or cytokine receptor- like factor 2 (CRLF2), forms a functional heterodimeric complex with IL-7 receptor alpha (IL-7Rα) to bind with TSLP. The present study aimed to elucidate the expression and epigenetic alterations of TSLP gene parallel to TSLP receptor and IL-10 genes expression in endometrial tissues of patients with endometriosis compared to controls. Materials & methodsIn this case-control study, 45 women with and without endometriosis was enrolled. The relative expression of TSLP, TSLPR and IL-10 genes were examined using qPCR. Chromatin Immunoprecipitation (ChIP) was also used to monitor epigenetic marks of methylation and acetylation on lysine 9 of histone H3 (H3K9me/ac) and DNA methylation in TSLP promoter. Results and conclusionTSLP, TSLPR and IL-10 genes were overexpressed in ectopic endometriotic lesions compared to controls. In ectopic samples, significant H3K9 hyper-acetylation and hypo-methylation parallel to DNA hypo-methylation were detected in TSLP promoter compared to eutopic and control groups (p < 0.05). These epigenetic changes were aligned with TSLP gene expression profile. These data collectively identify TSLP and TSLPR as candidate genes critically involved in development of endometriosis beyond their role in promoting Th2 immune responses. In addition, acetylation and methylation of H3K9 may have effective roles in TSLP dysregulation in endometriosis.

CRLF2 overexpression results in reduced B-cell differentiation and upregulated E2F signaling in the Dp16 mouse model of Down syndrome

Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia

AbstractThe process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3−/− preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

CRLF1 and CLCF1 in Development, Health and Disease

Cytokines and their receptors have a vital function in regulating various processes such as immune function, inflammation, haematopoiesis, cell growth and differentiation. The interaction between a cytokine and its specific receptor triggers intracellular signalling cascades that lead to altered gene expression in the target cell and consequent changes in its proliferation, differentiation, or activation. In this review, we highlight the role of the soluble type I cytokine receptor CRLF1 (cytokine receptor-like factor-1) and the Interleukin (IL)-6 cytokine CLCF1 (cardiotrophin-like cytokine factor 1) during development in physiological and pathological conditions with particular emphasis on Crisponi/cold-induced sweating syndrome (CS/CISS) and discuss new insights, challenges and possibilities arising from recent studies.

Prognostic significance of CRLF2 overexpression and JAK2 mutation in Egyptian pediatric patients with B-precursor acute lymphoblastic leukemia

BackgroundThe prognostic significance of cytokine receptor-like factor 2 (CRLF2) overexpression in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) is still controversial. We aimed to investigate the role of CRLF2 overexpression and JAK2 mutation in the diagnosis and prognosis of newly diagnosed pediatric B-ALL patients. MethodsCRLF2 expression was assessed by real-time quantitative polymerase chain reaction (PCR) in 115 pediatric patients newly diagnosed with precursor B-ALL patients compared with 24 age- and sex-matched controls. JAK2 R683G mutation status was performed by the qBiomarker Somatic Mutation PCR Assay. ResultsCRLF2 overexpression was identified in 21 patients (18.3%), while the JAK2 R683G mutant type was found in only in 7 patients (6.1%). There was a significant CRLF2 overexpression in patients with high initial TLC, high blast count in blood, and organomegaly (P .04, 0.005 & 0.05 respectively). No patients with CRLF2 overexpression expressed any recurrent cytogenetic translocations. 4 patients with CRLF2 overexpression showed JAK2 R683G mutation. CRLF2 levels and JAK2 R683G mutation status did not have a significant impact on either overall survival or disease-free survival. ConclusionCRLF2 expression was significantly higher in Egyptian precursor B-ALL pediatric patients. CRLF2 overexpression was associated with a number of unfavorable prognostic factors with high tumor load, but was not an adverse independent parameter in pediatric BCP-ALL patients. Some patients with CRLF2 overexpression display JAK2 mutation, which may benefit from targeted therapy by kinase inhibitors.

HMGN1 plays a significant role in CRLF2 driven Down Syndrome leukemia and provides a potential therapeutic target in this high-risk cohort.

The genetic basis of the predisposition for Down Syndrome (DS) patients to develop cytokine receptor-like factor 2 rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) is currently unknown. Genes located on chromosome 21 and expressed in hematopoietic cells are likely candidates for investigation of CRLF2r DS-ALL pathogenesis. We explored the high-mobility group nucleosome-binding protein 1 (HMGN1), located in the DS critical region, in an inducible CRISPR/Cas9 knockout (KO) xenograft model to assess the effect of HMGN1 loss of function on the leukemic burden. We demonstrated HMGN1 KO-mitigated leukemic phenotypes including hepatosplenomegaly, thrombocytopenia, and anemia, commonly observed in leukemia patients, and significantly increased survival in vivo. HMGN1 overexpression in murine stem cells and Ba/F3 cells in vitro, in combination with P2RY8-CRLF2, resulted in cytokine-independent transformation and upregulation of cell signaling pathways associated with leukemic development. Finally, in vitro screening demonstrated successful targeting of P2RY8-CRLF2 and HMGN1 co-expressing cell lines and patient samples with fedratinib (JAK2 inhibitor), and GSK-J4 (demethylase inhibitor) in combination. Together, these data provide critical insight into the development and persistence of CRLF2r DS-ALL and identify HMGN1 as a potential therapeutic target to improve outcomes and reduce toxicity in this high-risk cohort of young patients.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8–47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

Philadelphia-like acute lymphoblastic leukemia: Characterization in a pediatric cohort in a referral center in Colombia.

BackgroundPhiladelphia‐like (Ph‐like) acute lymphoblastic leukemia (ALL) is a subtype of pediatric leukemia with high risk factors and poor outcome. There are few reports of its prevalence in Latin America.AimThis study evaluated the frequency and clinical and biological characteristics of Ph‐like ALL in a pediatric cancer center in Colombia.MethodsThe Ph‐like genetic profile was analyzed by a low‐density array (LDA). Samples from patients with Ph‐like ALL were analyzed by fluorescent in situ hybridization for cytokine receptor like factor 2 (CRLF2) and ABL proto‐oncogene 1, non‐receptor tyrosine kinase (ABL1) rearrangements. Copy number variations were assessed by multiplex ligation probe amplification.ResultsData from 121 patients were analyzed. Fifteen patients (12.4%) had Ph‐like ALL, and these patients had significantly higher leukocyte counts at diagnosis and higher levels of minimal residual disease on days 15 and 33 of induction than patients without the Ph‐like subtype. There were no significant differences in sex, age, or response to prednisone at day 8 between the two groups. CRLF2 rearrangements were identified in eight patients, and ABL1 rearrangements were identified in two patients. Other genetic alterations alone or in combination were identified in 77% of patients, including deletions in cyclin dependent kinase inhibitor 2 A/B (46.2%), IKAROS family zinc finger 1 (38.3%), and paired box 5 (30.8%).ConclusionsPh‐like ALL had a 12.4% prevalence in our cohort of patients with pediatric ALL. The identification of this group of patients has importance for risk stratification and future targeted therapy.

Precision Co-Targeting of the Thymic Stromal Lymphopoietin Receptor in Childhood CRLF2-Rearranged Acute Lymphoblastic Leukemia

Introduction : Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of chemoresistance and relapse. CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Ph-like and 60% of Down Syndrome (DS)-associated ALL and induce constitutive JAK/STAT and other kinase signaling. Current clinical trials are studying chemotherapy with the JAK inhibitor ruxolitinib in patients with CRLF2-rearranged Ph-like ALL, but results are not yet known. While chimeric antigen receptor T-cell (CART) immunotherapies have induced remarkable remissions in children with relapsed/refractory B-ALL, approximately 50% of CD19CART-treated patients will relapse again, many with CD19 antigen loss. New therapies are needed to prevent relapse and overcome immunotherapeutic resistance.Methods : We previously developed CAR T cells targeting the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) and demonstrated potent preclinical activity in Ph-like ALL models (Qin Blood 2015), which has led to a soon-to-open phase 1 clinical trial for patients with relapsed/refractory CRLF2-overexpressing ALL. In the current preclinical studies, we hypothesized that combinatorial targeting with bispecific TSLPRxCD19CART or TSLPRxCD22CART (Ross Cancer Res 2020) or with TSLPRCART + ruxolitinib will have superior activity against CRLF2-rearranged Ph-like and DS-ALL.Results : TSLPRCART treatment of CRLF2-rearranged ALL cell line (n=1) and patient-derived xenograft (PDX) models potently inhibited leukemia proliferation in vitro and in vivo and induced long-term ‘cure’ of xenograft mice. However, co-administration of TSLPRCART + ruxolitinib markedly diminished in vivo T cell numbers, blunted cytokine production, and facilitated leukemia relapse, which could be abrogated by delaying ruxolitinib. Importantly, ruxolitinib co-treatment prevented severe TSLPRCART-induced cytokine release syndrome (CRS) and animal death. Interestingly, ruxolitinib withdrawal led to return of T-cell functionality with re-detection of TSLPRCART in peripheral blood, induction of IFN-γ production, and leukemia clearance upon CRLF2+ ALL rechallenge (Figure 1).Conclusions: In these preclinical studies, we report potent activity of TSLPRCART in cell line (n=1) and PDX models of childhood CRLF2-rearranged Ph-like ALL (n=2) and DS-ALL (n=2) and, interestingly, deleterious effects of concomitant JAK inhibition upon CAR T cell functionality. We demonstrated that ruxolitinib co-administration impaired in vivo TSLPRCART-induced ALL cell killing but was also beneficial in protection against life-threatening cytokine release syndrome in co-treated animals. Importantly, TSLPRCART was not eliminated, only suppressed, by JAKi co-treatment with restoration of T cell functionality upon ruxolitinib removal and/or leukemia relapse/rechallenge studies. Ongoing studies are defining optimal TSLPRCART + ruxolitinib sequence(s) to maximize both anti-leukemia efficacy and potential CRS mitigation, as well as assessing in vivo efficacy of bispecific TSLPRCARTs in CRLF2-R Ph-like ALL and DS-ALL PDX models for future translation and clinical evaluation in next-generation trials. [Display omitted] DisclosuresFry: ElevateBio: Research Funding; Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tasian: Aleta Biotherapeutics: Consultancy; Kura Oncology: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

MiR-3065-3p promotes stemness and metastasis by targeting CRLF1 in colorectal cancer

BackgroundColorectal cancer is one of the most common malignancy in the world. It has been reported that cancer stem cells (CSCs) serve as the primary drivers of tumorigenesis and tumor progression. There is an urgent need to explore novel molecules that regulate CSCs or their signatures. Increasing evidence has shown that miRNAs are involved in tumorigenesis and progression. Here, we aim to explore the regulatory effect and mechanism of miR-3065-3p on the stemness of colorectal cancer.MethodsThe expression of miR-3065-3p in colorectal cancer and the association of miR-3065-3p expression with prognosis of patients with colorectal cancer were analyzed using TCGA dataset or clinical cases. Gain or loss of function in different models, including colorectal cancer cell lines and orthotopic xenograft or liver metastatic mouse model, were used to investigate the effects of miR-3065-3p on colorectal cancer stemness and metastasis in vitro and in vivo. Cancer stemness was analyzed by detecting the ability of migration and invasion, NANOG, OCT4, and SOX2 expression, ALDH activity and sphere formation. In addition, the interaction of miR-3065-3p and cytokine receptor-like factor 1 (CRLF1) was analyzed theoretically and identified by the luciferase reporter assay. Moreover, the correlation between CRLF1 expression and miR-3065-3p was analyzed in colorectal cancer tissues. Finally, the effect of CRLF1 on the stemness and metastasis of colorectal cancer in vitro and in vivo was assessed.ResultsIn this report, we found that miR-3065-3p was overexpressed in colorectal cancer and that its high expression was associated with poor prognosis of patients with colorectal cancer. miR-3065-3p promotes the stemness and metastasis of colorectal cancer. Furthermore, CRLF1 was the downstream target of miR-3065-3p and inhibited the stemness of colorectal cancer. In addition, CRLF1 expression was negatively correlated with miR-3065-3p in colorectal cancer tissues. And, CRLF1 mediated the effects of miR-3065-3p on promoting stemness of colorectal cancer cells.ConclusionOur data suggest that miR-3065-3p promoted the stemness and metastasis of colorectal cancer by targeting CRLF1. miR-3065-3p might serve as a promising prognostic marker as well as a therapeutic target for colorectal cancer.

Suppression of CRLF1 promotes the chondrogenic differentiation of bone marrow-derived mesenchymal stem and protects cartilage tissue from damage in osteoarthritis via activation of miR-320

BackgroundOsteoarthritis (OA) is the most prevalent chronic joint disease, and is hard to be cured at present. Cytokine receptor-like factor 1 (CRLF1) has been identified as an upregulated gene in OA cartilage. However, the precise identities and functions of CRLF1 in OA progression have remained to be fully elucidated.MethodsWe used a murine model of injury-induced OA (destabilization of medial meniscus, DMM) and BMSCs to investigate the specific biological functions and mechanisms of CRLF1.ResultsWe found that CRLF1 was significantly increased in the DMM surgery-induced OA model and was down-regulated during chondrogenic differentiation of BMSCs. Luciferase reporter assays showed that CRLF1 was a direct target of miR-320 in BMSCs. miR-320 can reverse the effect of CRLF1 on cell proliferation, apoptosis and chondrogenic differentiation of BMSCs. Furthermore, knockdown of CRLF1 or over-expression of miR-320 can inhibit the apoptosis of primary chondrocytes.ConclusionSuppression of CRLF1 promotes the chondrogenic differentiation of BMSCs and protects cartilage tissue from damage in osteoarthritis via activation of miR-320.

Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia

A recombinant antibody fragment directed to the thymic stromal lymphopoietin receptor (CRLF2) efficiently targets pediatric Philadelphia chromosome-like acute lymphoblastic leukemia

Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.

SUMMARYNeurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.