Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

Identification of putative genetic variants in major depressive disorder patients in Pakistan.

BACKGROUND: Major depressive disorder (MDD) is a polygenic, and highly prevalent disorder affecting 322 million people globally. It results in several psychological changes which adversely affect different dimensions of life and may lead to suicide. METHODS: Whole exome sequencing of 15 MDD patients, enrolled at the Dr. A. Q. Khan Institute of Behavioral Sciences, Karachi, was performed using NextSeq500. Different bioinformatics tools and databases like ANNOVAR, ALoFT, and GWAS were used to identify both common and rare variants associated with the pathogenesis of MDD. RESULTS: A total of 1985 variations were identified in 479 MDD-related genes. Several SNPs including rs1079610, rs11750538, rs1799913, rs1801131, rs2230267, rs2231187, rs3819976, rs4314963, rs56265970, rs587780434, rs6330, rs75111588, rs7596487, and rs9624909 were prioritized due to their deleteriousness and frequency difference between the patients and the South Asian population. A non-synonymous variation rs56265970 (BCR) had 26% frequency in patients and was not found in the South Asian population; a multiallelic UTR-5' insertion rs587780434 (RELN) was present with an allelic frequency of 70% in patients whereas 22% in the SAS population. Genetic alterations in PABPC1 genes, a stress-associated gene also had higher allele frequency in the cases than in the normal population. CONCLUSION: This present study identifies both common and rare variants in the genes associated with the pathogenesis of MDD in Pakistani patients. Genetic variations in BCR, RELN, and stress-associated PABPC1 suggest potential roles in the pathogenesis of MDD.

DDR1 contributes to kidney inflammation and fibrosis by promoting the phosphorylation of BCR and STAT3.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease. However, its role in acute kidney injury and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia/reperfusion-induced acute kidney injury that progressed to kidney fibrosis in WT and Ddr1-null mice. We showed that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increased ß-catenin activity and in turn MCP-1 production. Finally, we showed that DDR1-mediated STAT3 activation was required to stimulate the secretion of TGF-ß. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-ß. These findings identify DDR1 an attractive therapeutic target for ameliorating both proinflammatory and profibrotic signaling in kidney disease.

A rare case of atypical chronic myeloid leukemia associated with t(8;22)(p11.2;q11.2)/ BCR-FGFR1 rearrangement: A case report and literature review.

Myeloid/lymphoid neoplasm with t(8;22)(p11.2;q11.2)/BCR-FGFR1 is an extremely rare diagnosis, with few reported cases to date. In contrast to other FGFR1-partner rearrangements that are associated with chronic eosinophilic leukemia, acute myeloid leukemia, and/or lymphoblastic lymphoma, patients with BCR-FGFR1 have a myeloproliferative disorder that closely resembles chronic myeloid leukemia (CML). The current report describes a rare case of a 61 year old man with an atypical CML phenotype associated with t(8;22)(p11.2;q11.2)/BCR-FGFR1. A literature review is presented to enhance the awareness of this rare diagnostic entity.

A case of a patient characterized by t(8;22)(p11;q11) and BCR/FGFR1 fusion gene, who was successfully treated with haploidentical hematopoietic stem cell transplantation.

Objective: The 8p11 myeloproliferative syndrome [EMS] is a rare myeloproliferative disorder which usually develops rapidly with chromosomal translocation of the fibroblast growth factor receptor 1 gene. The gene has 15 fusion partners, including the breakpoint cluster region (BCR) gene on chromosome 22. Of all the tests available, chromosome karyotype determination is the most important for the diagnosis of EMS. Here, we describe one case of a patient characterized by marked increase of white blood cells and thrombocytopenia and diagnosed as EMS with t(8;22)(p11;q11) by chromosome karyotype.Methods: 28-year-old man was referred to our hospital. He had a onemonth history of intermittent coughing and a small amount of expectoration after catching a cold. As an outpatient, his complete blood count showed: WBC was 130.04 × 109/L with 80.20% granulocytes.Hematologic investigations, bone marrow analysis and genomic DNA sequencing studies were performed.Results: Despite additional chromosomal abnormalities,the patient progressed rapidly with a B blast cell clone in one month. After diagnosis inthree months, the patient underwent the haplo-identical BMT of his brother, followed up for three years, and had a high rate of survival.Conclusions: Our report provides a definite conceptual framework for a better understanding of the characteristics of The 8p11 myeloproliferative syndrome [EMS].

Genomics of Resistance to Targeted Therapies.

Targeting BCR and BCL-2 signaling is a widely used therapeutic strategy for chronic lymphocytic leukemia. C481S mutation decreases the covalent binding affinity of ibrutinib to BTK, resulting in reversible rather than irreversible inhibition. In addition to BTK, mutations in PLCG2 have been demonstrated to mediate acquired ibrutinib resistance. Venetoclax, a highly selective BCL2 inhibitor, has high affinity to the BH3-binding grove of BCL2. Mutation in BCL2 (Gly101Val) decreases the affinity of BCL2 for venetoclax and confers acquired resistance in cell lines and primary patient cells. This review discusses the common mechanisms of resistance to targeted therapies in chronic lymphocytic leukemia.

Loss function of Bcr mutation causes gastrointestinal dysmotility and brain developmental defects.

BACKGROUND: The breakpoint cluster region (BCR) is a protein that originally forms a fusion protein with c-Abl tyrosine kinase and induces leukemia. Researchers have shown that BCR is enriched in the central nervous system and may contribute to neurological disorders. We aimed to investigate the physiological function of BCR in neural development in the gastrointestinal (GI) tract and brain. METHODS: Whole-exome sequencing was used to screen for mutations in the BCR. Bcr knockout mice (Bcr-/- , ΔExon 2-22) were generated using the CRISPR/Cas9 system. Transit of carmine red dye and glass bead expulsion assays were used to record total and proximal GI transit and distal colonic transit. KEY RESULTS: In an infant with pediatric intestinal pseudo-obstruction, we found a heterozygous de novo mutation (NM_004327.3:c.3072+1G>A) in BCR. Bcr deficiency mice (Bcr-/- ) exhibited growth retardation and impaired gastrointestinal motility. Bcr-/- mice had a prolonged average total GI transit time with increased distal colonic transit and proximal GI transit in isolation. Morphology analysis indicated that Bcr-/- mice had a less number of neurons in the submucosal plexus and myenteric plexus. Bcr-/- mice exhibited apparent structural defects in the brain, particularly in the cortex. Additionally, Bcr- depletion in the mouse cortex altered the expression of Ras homologous (Rho) family small GTPases. CONCLUSIONS AND INFERENCES: BCR mutations are associated with intestinal obstruction in children. Loss of Bcr can cause intestinal dysmotility and brain developmental defects may via regulation of Rho GTPases.

Molecular classification improves risk assessment in adult BCR-ABL1-negative B-ALL.

Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1- B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5-year OS of 0% to 27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); 20.3% had intermediate-risk genotypes with 5-year OS of 33% to 45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like, and TP53 mutations in patients who were low-hypodiploid (54%) and BCL2/MYC-rearranged (33%) but were not independently associated with outcome. Of patients considered high risk based on presenting age and white blood cell count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like, and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non-risk-adapted approach on a single trial show the prognostic importance of genomic analyses, which may translate into future therapeutic benefits.

BAFF promotes heightened BCR responsiveness and manifestations of chronic GVHD after allogeneic stem cell transplantation.

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic µMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.

Identification of "regulation of RhoA activity panel" as a prognostic and predictive biomarker for gastric cancer.

RhoA is a member of the RHO family GTPases and is associated with essential functions in gastric cancer. In this study, we identified a gastric cancer biomarker, termed the "regulation of RhoA activity panel" (RRAP). Patients with gastric cancer from The Cancer Genome Atlas database were divided into training (N=160) and validation (N=155) cohorts. A cohort of 109 Chinese gastric cancer patients was utilized as an independent validation. Patients with mutated RRAP showed significantly better overall survival than patients with wild type RRAP. We also analyzed the association between RRAP and the migration capacity, immune-related signatures, and the tumor microenvironment. RRAP-mutant tumors had a significantly lower degree of lymph node metastasis and lower activities of migration-related pathways. These tumors also showed significantly increased immune cell infiltration and cytotoxic activity. Furthermore, two independent patient cohorts who received immune checkpoint blockade therapy were assessed for RRAP mutant status. As expected, for both immunotherapy cohorts, higher response rates to immune checkpoint blockade therapy were observed in patients with RRAP-mutant tumors than in patients with wild type RRAP tumors. Overall, this study indicates that the RRAP gene set is a potential biomarker for gastric cancer prognosis and therapeutic selection.

Integrated genomic analysis using chromosomal microarray, fluorescence in situ hybridization and mate pair analyses: Characterization of a cryptic t(9;22)(p24.1;q11.2)/BCR-JAK2 in myeloid/lymphoid neoplasm with eosinophilia.

The 2016 World Health Organization entity 'Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2' encompasses a group of rare neoplasms that result from the formation of a fusion gene that leads to expression of an aberrant tyrosine kinase. This entity also contains variant JAK2 fusion partners, and detection of this defining event can be facilitated by various cytogenetic and molecular methods. Cryptic rearrangements of 9p24/JAK2 can be particularly challenging to identify. We describe the use of chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) with a probe for JAK2, and genomic mate pair analysis to describe a complex karyotype with a t(9;22) that produced a functional BCR-JAK2 fusion, leading to the appropriate diagnosis for the patient. This case highlights the importance of using an integrated genomic approach to fully define complex aberrations to assign proper diagnoses.

Spectrum of gene mutations identified by targeted next-generation sequencing in Chinese leukemia patients.

BACKGROUND: Despite targeted sequencing have identified several mutations for leukemia, there is still a limit of mutation screening for Chinese leukemia. Here, we used targeted next-generation sequencing for testing the mutation patterns of Chinese leukemia patients. METHODS: We performed targeted sequencing of 504 tumor-related genes in 109 leukemia samples to identify single-nucleotide variants (SNVs) and insertions and deletions (INDELs). Pathogenic variants were assessed based on the American College of Medical Genetics and Genomics (ACMG) guidelines. The functional impact of pathogenic genes was explored through gene ontology (GO), pathway analysis, and protein-protein interaction network in silico. RESULTS: We identified a total of 4,655 SNVs and 614 INDELs in 419 genes, in which PDE4DIP, NOTCH2, FANCA, BCR, and ROS1 emerged as the highly mutated genes. Of note, we were the first to demonstrate an association of PDE4DIP mutation and leukemia. Based on ACMG guidelines, 39 pathogenic and likely pathogenic mutations in 27 genes were found. GO annotation showed that the biological process including gland development, leukocyte differentiation, respiratory system development, myeloid leukocyte differentiation, mesenchymal to epithelial transition, and so on were involved. CONCLUSION: Our study provided a map of gene mutations in Chinese patients with leukemia and gave insights into the molecular pathogenesis of leukemia.

Dual-modal label-free genosensor based on hemoglobin@gold nanocluster stabilized graphene nanosheets for the electrochemical detection of BCR/ABL fusion gene.

For the first time, we have successfully synthesized stable graphene nanosheets from graphite powder through sonication in the hemoglobin-capped gold nanoclusters (Hb@AuNCs) solution for biosensing application. This approach, as a simple method for the exfoliation and fragmentation of graphite in a nanocluster solution, enabled us to produce stable aqueous graphene dispersions at low cost and without the need for hazardous chemicals or tedious experimental procedures. In this method, Hb@AuNCs were used not only as stabilizing agent of graphene through non-covalent bonding, but also as dispersing agent of few-layer graphene nanosheets. The Hb@AuNCs stabilized graphene (Hb@AuNCs-G) was characterized by high resolution transmission electron microscopy (HRTEM), zeta-sizer and Raman spectroscopy. Then, the graphene nanosheets were applied as a novel versatile electrochemical platform for ultrasensitive biosensing of short DNA species of chronic myelogenous leukemia (CML) based on the "signal off" and "signal on" strategies. For this purpose, a single strand DNA (ssDNA) was immobilized on the Hb@AuNCs-G/AuNPs modified electrode surface and acted as the biorecognition element. Methylene blue (MB), as the signaling probe, was then intercalated into the ssDNA. The intercalated MB was liberated upon interaction with the synthetic complementary DNA (cDNA, target), thereby resulting in the apparent reduction of MB redox signal. This designed "signal off" sensing system enabled the voltammetric determination of the target cDNA over a dynamic linear range (DLR) of 0.1 fM to 10 pM with a limit of detection (LOD) of 0.037 fM. In the "signal on" strategy, the response to the cDNA was detected by monitoring the change in the electron transfer resistance (Rct) using the ferro/ferricyanide system as a redox probe. The charge transfer resistance of the probe was found to increase linearly with increasing concentration of target cDNA in the range of 0.1 fM-10 pM with a limit of detection of 0.030 fM. Finally, the selectivity and feasibility of genosensor was evaluated by the analysis of derived nucleotides from mismatched sequences and the clinical samples of patients with leukemia as real samples, respectively.

CRISPR/CAS9-mediated knockout of Abi1 inhibits p185Bcr-Abl-induced leukemogenesis and signal transduction to ERK and PI3K/Akt pathways.

BACKGROUND: Abl interactor 1 (Abi1) is a downstream target of Abl tyrosine kinases and a component of the WAVE regulatory complex (WRC) that plays an important role in regulating actin cytoskeleton remodeling and membrane receptor signaling. While studies using short hairpin RNA (shRNA) have suggested that Abi1 plays a critical role in Bcr-Abl-induced leukemogenesis, the mechanism involved is not clear. METHODS: In this study, we knocked out Abi1 expression in p185Bcr-Abl-transformed hematopoietic cells using CRISPR/Cas9-mediated gene editing technology. The effects of Abi1 deficiency on actin cytoskeleton remodeling, the Bcr-Abl signaling, IL-3 independent growth, and SDF-induced chemotaxis in these cells were examined by various in vitro assays. The leukemogenic activity of these cells was evaluated by a syngeneic mouse transplantation model. RESULTS: We show here that Abi1 deficiency reduced the IL3-independent growth and SDF-1α-mediated chemotaxis in p185Bcr-Abl-transformed hematopoietic cells and inhibited Bcr-Abl-induced abnormal actin remodeling. Depletion of Abi1 also impaired the Bcr-Abl signaling to the ERK and PI3 kinase/Akt pathways. Remarkably, the p185Bcr-Abl-transformed cells with Abi1 deficiency lost their ability to develop leukemia in syngeneic mice. Even though these cells developed drug tolerance in vitro after prolonged selection with imatinib as their parental cells, the imatinib-tolerant cells remain incapable of leukemogenesis in vivo. CONCLUSIONS: Together, this study highlights an essential role of Abi1 in Bcr-Abl-induced leukemogenesis and provides a model system for dissecting the Abi1 signaling in Bcr-Abl-positive leukemia.

Prognostic Significance of bcr-1 and bcr-3 Isoforms of PML-RARA and FLT3-ITD in Patients With Acute Promyelocytic Leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) has a characteristic peculiar morphologic and genetic features as well as a more favorable outcome. We studied the differential effect of bcr-1 and bcr-3 isoforms of the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) transcript together with FLT3 internal tandem duplication (FLT3-ITD) mutation status on the outcome of newly diagnosed de novo APL patients. PATIENTS AND METHODS: This cohort study included all patients diagnosed with APL at outpatient medical and pediatric oncology clinics of the National Cancer Institute, Cairo University, Cairo, Egypt, from May 2012 to January 2018. RESULTS: The study included 118 patients with APL, 71 adults (60.2%) and 47 children (39.8%). Median (range) age was 25 (1.5-70) years. Children had significantly higher total leukocyte count (≥10 × 109/L), disseminated intravascular coagulation (DIC), and thrombocytopenia (< 40 × 109/L) than adults (P = .04, .03, and .04, respectively), while the latter group had significantly higher hemorrhage than children (P = .04). FLT3-ITD mutation was detected in the whole group, children, and adults in 23.7%, 30.6%, and 24.6%, respectively. FLT3-ITD mutation was significantly associated with leukocytosis in the whole group (P = .039). bcr-3 was significantly associated with FLT3-ITD mutation in the whole APL cohort and in adults (P = .011, P = .022). All children (both bcr-1 and bcr-3) and all adult patients with bcr-3 experienced CR, while 22 (78.5%) of 28 patients with bcr-1 experienced CR (P = .04). APL patients with DIC and hemorrhage had significantly lower overall survival (P = .002 and < .001, respectively). Overall survival for APL in children was significantly better than in adults (P = .005). Multivariate analysis indicated that age was an independent prognostic variable affecting survival (hazard ratio = 2.6; 95% confidence interval, 1.3-5.3; P = .007) (adults had hazard ratio of death 2.6 times higher than children). DIC and FLT3-ITD were independent prognostic variables affecting survival in children with APL (hazard ratio = 12.3; 95% confidence interval, 1.46-104.61; P = .021; and hazard ratio = 5.2; 95% confidence interval, 1.01-26.95; P = .048, respectively). CONCLUSION: Age is an independent prognostic factor for APL. bcr-3 is significantly associated with FLT3-ITD in adults with APL. DIC and FLT3-ITD are adverse prognostic factors in children with APL. Despite children being at higher risk, outcome is better than in adults.

Functional characterization of two rare BCR-FGFR1+ leukemias.

8p11 myeloproliferative syndrome (EMS) represents a unique World Health Organization (WHO)-classified hematologic malignancy defined by translocations of the FGFR1 receptor. The syndrome is a myeloproliferative neoplasm characterized by eosinophilia and lymphadenopathy, with risk of progression to either acute myeloid leukemia (AML) or T- or B-lymphoblastic lymphoma/leukemia. Within the EMS subtype, translocations between breakpoint cluster region (BCR) and fibroblast growth factor receptor 1 (FGFR1) have been shown to produce a dominant fusion protein that is notoriously resistant to tyrosine kinase inhibitors (TKIs). Here, we report two cases of BCR-FGFR1+ EMS identified via RNA sequencing (RNA-seq) and confirmed by fluorescence in situ hybridization (FISH). Sanger sequencing revealed that both cases harbored the exact same breakpoint. In the first case, the patient presented with AML-like disease, and in the second, the patient progressed to B-cell acute lymphoblastic leukemia (B-ALL). Additionally, we observed that that primary leukemia cells from Case 1 demonstrated sensitivity to the tyrosine kinase inhibitors ponatinib and dovitinib that can target FGFR1 kinase activity, whereas primary cells from Case 2 were resistant to both drugs. Taken together, these results suggest that some but not all BCR-FGFR1 fusion positive leukemias may respond to TKIs that target FGFR1 kinase activity.

Critical individual roles of the BCR and FGFR1 kinase domains in BCR-FGFR1-driven stem cell leukemia/lymphoma syndrome.

Constitutive activation of FGFR1, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The BCR-FGFR1 variant is unique in that the BCR component contributes a serine-threonine kinase (STK) to the N-terminal end of the chimeric FGFR1 kinase. We have deleted the STK domain and mutated the critical Y177 residue and demonstrate that the transforming activity of these mutated genes is reduced compared to the BCR-FGFR1 parental kinase. In addition, we demonstrate that deletion of the FGFR1 tyrosine kinase domain abrogates transforming ability, which is not compensated for by BCR STK activity. Unbiased screening for proteins that are inactivated as a result of loss of the BCR STK identified activated S6 kinase and SHP2 kinase. Genetic and pharmacological inhibition of SHP2 function in SCLL cells expressing BCR-FGFR1 in vitro leads to reduced viability and increased apoptosis. In vivo treatment of SCLL in mice with SHP099 leads to suppression of leukemogenesis, supporting an important role for SHP2 in FGFR1-driven leukemogenesis. In combination with the BGJ398 FGFR1 inhibitor, cell viability in vitro is further suppressed and acts synergistically with SHP099 in vivo suggesting a potential combined targeted therapy option in this subtype of SCLL disease.

Oncogenic fusion protein BCR-FGFR1 requires the breakpoint cluster region-mediated oligomerization and chaperonin Hsp90 for activation.

Mutation and translocation of fibroblast growth factor receptors often lead to aberrant signaling and cancer. This work focuses on the t(8;22)(p11;q11) chromosomal translocation which creates the breakpoint cluster region (BCR) fibroblast growth factor receptor1 (FGFR1) (BCR-FGFR1) fusion protein. This fusion occurs in stem cell leukemia/lymphoma, which can progress to atypical chronic myeloid leukemia, acute myeloid leukemia, or B-cell lymphoma. This work focuses on the biochemical characterization of BCR-FGFR1 and identification of novel therapeutic targets. The tyrosine kinase activity of FGFR1 is required for biological activity as shown using transformation assays, interleukin-3 independent cell proliferation, and liquid chromatography/mass spectroscopy analyses. Furthermore, BCR contributes a coiled-coil oligomerization domain, also essential for oncogenic transformation by BCR-FGFR1. The importance of salt bridge formation within the coiled-coil domain is demonstrated, as disruption of three salt bridges abrogates cellular transforming ability. Lastly, BCR-FGFR1 acts as a client of the chaperonin heat shock protein 90 (Hsp90), suggesting that BCR-FGFR1 relies on Hsp90 complex to evade proteasomal degradation. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor Ganetespib, and also respond to combined treatment with Ganetespib plus the FGFR inhibitor BGJ398. Collectively, these data suggest novel therapeutic approaches for future stem cell leukemia/lymphoma treatment: inhibition of BCR oligomerization by disruption of required salt bridges; and inhibition of the chaperonin Hsp90 complex.