Wild-type PAX5 Research Articles

Familial Predisposition to B-Cell Precursor Acute Lymphoblastic Leukemia Mediated By PAX5 Germline Variants

Introduction: Somatic PAX5 alterations are one of the most common features present in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Recently, PAX5alt and PAX5 p.P80R have been identified as two novel PAX5-driven BCP-ALL subtypes (Gu et al., Nat Genet., 2019). PAX5 is a well-known master regulator for B-cell development, hence, deregulated PAX5 expression poses high risks for leukemia development. In this regard, families with germline PAX5 variants are a unique source to study the effect of PAX5 on leukemia development in humans. Methods: We corroborated clinical data with immunophenotyping and genomic analyses of families carrying PAX5 germline variants. Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 who developed BCP-ALL, including one family with a so far undescribed amino acid substitution p.Gly183Arg. To identify additional synergizing germline variants, whole exome sequencing of both families, together with one family previously reported by Auer et al., (Leukemia, 2014) was carried out. This analysis yielded no common germline variants apart from PAX5, which could explain the incomplete penetrance in the observed leukemia development, but confirmed the families’ diverse backgrounds in different ancestry. Interestingly, an in-depths B-cell differentiation study comparing PBMCs from PAX5 variant carriers (n=8) in the respective families and unrelated non-carriers (n=44) revealed a significant decrease of intermittent B-cells (Student's T-test, 5.796% vs 9.820%, p= 0.017) and memory B-cells (Mann-Whitney U, 10.760% vs 17.350%, p= 0.003) in carriers versus non-carriers. Moreover, bone marrow analysis from one of the patients carrying PAX5 p.G183S showed a reduction in the number of mature B cells, suggesting deregulation of the B-cell differentiation at the pre-B stage. Altogether, a total of 21 patients from seven families carrying three different heterozygous germline PAX5 variants have been reported. Statistically significant differences are observed between the age of onset of patients harboring the PAX5 p.Arg38His variant compared with patients carrying p.Gly183Ser/Arg (Student's T-test, 17.75 vs. 2.70 years old, respectively; p=0.03). Furthermore, all patients who developed BCP-ALL showed somatic chromosome 9p alterations with loss of heterozygosity of the Wildtype PAX5 locus. Concordantly, all families display incomplete penetrance, with PAX5 p.Gly183Arg and p.Arg38His being by far more penetrant than p.Gly183Ser. Regarding the outcome, a high rate of overall survival (72%) and a high rate of relapse (75%) were observed. 54% of the patients received hematopoietic stem cell transplantation, and of these, 71% survived. Conclusion: Taken together, our data provide evidence for deregulated B-cell development mediated by reduced PAX5 levels in the germline of families carrying PAX5 germline variants. Our results emphasize the benefit of PAX5 germline variant screening in patients diagnosed with BCP-ALL and somatic alterations on chromosome 9p, regardless of a family history of leukemia.

Characterization of Novel Subtypes in B Progenitor Acute Lymphoblastic Leukemia

▪IntroductionB progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor.MethodsTo identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing.ResultsUsing integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in “double/triple hit” lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion.We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL.ConclusionsThese results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. DisclosuresMcKay:ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations.

PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.

Loss of Pax5 Exploits Sca1-BCR-ABLp190 Susceptibility to Confer the Metabolic Shift Essential for pB-ALL.

Preleukemic clones carrying BCR-ABLp190 oncogenic lesions are found in neonatal cord blood, where the majority of preleukemic carriers do not convert into precursor B-cell acute lymphoblastic leukemia (pB-ALL). However, the critical question of how these preleukemic cells transform into pB-ALL remains undefined. Here, we model a BCR-ABLp190 preleukemic state and show that limiting BCR-ABLp190 expression to hematopoietic stem/progenitor cells (HS/PC) in mice (Sca1-BCR-ABLp190) causes pB-ALL at low penetrance, which resembles the human disease. pB-ALL blast cells were BCR-ABL-negative and transcriptionally similar to pro-B/pre-B cells, suggesting disease onset upon reduced Pax5 functionality. Consistent with this, double Sca1-BCR-ABLp190+Pax5+/- mice developed pB-ALL with shorter latencies, 90% incidence, and accumulation of genomic alterations in the remaining wild-type Pax5 allele. Mechanistically, the Pax5-deficient leukemic pro-B cells exhibited a metabolic switch toward increased glucose utilization and energy metabolism. Transcriptome analysis revealed that metabolic genes (IDH1, G6PC3, GAPDH, PGK1, MYC, ENO1, ACO1) were upregulated in Pax5-deficient leukemic cells, and a similar metabolic signature could be observed in human leukemia. Our studies unveil the first in vivo evidence that the combination between Sca1-BCR-ABLp190 and metabolic reprogramming imposed by reduced Pax5 expression is sufficient for pB-ALL development. These findings might help to prevent conversion of BCR-ABLp190 preleukemic cells.Significance: Loss of Pax5 drives metabolic reprogramming, which together with Sca1-restricted BCR-ABL expression enables leukemic transformation. Cancer Res; 78(10); 2669-79. ©2018 AACR.

Transcription Factor Pax5 Promotes B-lymphomagenesis in a Promoter-Independent Manner

To determine whether B lymphocyte-specific transcription factor Pax5 regulates B-lympho-magenesis without direct binding to promoter. Mouse B-lymphoma cell line myc3 and 38B9 were infected with GFP- tagged retrovirus that encodes wide type or various mutant pax5 genes. After viral infection for 48 hours, the percentage of GFP positive lymphoma cells was determined by flow cytomety. The percentage of GFP positive tumor cells was further monitored every 3 days in vitro or once the tumor was formed in vivo. Both cell cycle and apoptic cell number of GFP positive lymphoma cells were analyzed using flow cytometry. Similar to the infection with wild type Pax5 retrovirus, infection with Pax5 mt 1-357 and Pax5 mt 304-358 that lacks of DNA binding motif can strongly increase the percentage of GFP+ B-lymphoma cells both in vitro and in vivo (P<0.01), while infection with empty viral vector migR-GFP and Pax5 mt 1-143 containing only DNA binding motif failed to increase the percentage of GFP positive tumor cells (P>0.05). Moreover, the analysis of flow cytometry demonstrated that more B-lymphoma cells infected with wild type Pax5, Pax5 mt 1-357 and Pax5 mt 304-358 retroviruses entered S and G2/M phases in comparison with those infected with empty viral vector migR-GFP and Pax5 mt 1-143. Apoptotic rates among different groups were not significantly changed. Pax5 can promote B-lymphoma cell growth both in vitro and in vivo in a promoter-independent manner. This is mainly due to the accelerating of cell cycle rather than decreasing apoptosis. Our studies provide potential theory for restraing B-lymphomagenesis by targeting the specific Pax5 domains.

Abstract 93: Transcriptional control of glucocorticoid responses in leukemia

Abstract Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. Transcriptional control of glucocorticoid responses in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 93. doi:10.1158/1538-7445.AM2017-93

Transcriptional Control of Glucose and Energy Supply Prevents Oncogenic Signaling and B Cell Transformation

Background and Hypothesis: B-cell identity is determined by a set of B-cell transcription factors including PAX5, IKZF1 and EBF1. However, B-lineage leukemia clones often carry secondary genetic lesions that result in reduced activity or inactivation of these transcription factors. Studying patient samples from clinical trials for B-lineage childhood (P9906; n=187) and adult (MDACC; n=92) leukemia, we found that genetic defects in one or more B-cell transcription factors represent near-obligate lesions in human acute lymphoblastic leukemia (209 of 279 B-lineage ALL cases). While previously of unknown significance, we found that adult ALL cases with known lesions in one or more of these transcription factors had higher activity of lactate dehydrogenase, and phospho-states indicated higher activity of IRS1, PDK1, and AKT, which contribute to glucose uptake. For this reason, we investigated whether B-cell transcription factors set metabolic constraints of oncogenic signaling in leukemia.Results: Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells carrying PAX5 and IKZF1deletions decreased phospho-AKT levels. Furthermore, protein levels of multiple positive regulators involved in glucose uptake and metabolism (including insulin receptor, glucose transporters and hexokinases) were downregulated upon reconstitution of PAX5 and IKZF1. Conversely, protein levels of negative regulators of glucose uptake and metabolism including NR3C1 and TXNIP were upregulated upon reinstatement of PAX5 and IKZF1 function. Analysis of ChIPseq data of human B cells revealed binding of multiple B-cell transcription factors including PAX5, IKZF1 and EBF1 to promoter regions of genes encoding positive regulators of glucose uptake and metabolism. Binding peaks for B-cell transcription factors were also observed at genes that encode negative regulators of glucose uptake and metabolism (NR3C1 and TXNIP). In addition, direct recruitment of PAX5 was confirmed by single-locus quantitative chromatin immunoprecipitation (qChIP).Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells caused depletion from the cell culture in competitive growth assays, in parallel with reduced glucose consumption and depletion of cellular ATP levels. On the contrary, overexpression of dominant-negative PAX5-ETV6 and IK6 in patient-derived B-lineage ALL cells expressing wildtype PAX5 and IKZF1 resulted in a net survival advantage, concomitant with increases in both glycolytic activity and cellular ATP levels. PAX5-mediated impaired survival fitness was significantly rescued by CRISPR-based activation of gene expression of insulin receptor, glucose transporter and hexokinases in patient-derived B-lineage ALL cells. Conversely, CRISPR/Cas9-mediated deletion of NR3C1 and TXNIP largely reversed the effects of PAX5. Finally, reduced survival fitness upon reconstitution of wild-type PAX5 and IKZF1 in patient-derived pre-B ALL cells was mostly rescued by metabolites that can enter the TCA cycle and thus provide ATP.Conclusions: In summary,B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation. Our findings suggest a causative link between impaired glucose uptake and metabolism caused by B-cell transcription factors as well as cell death, and the known tumor suppressive function of PAX5 and IKZF1 in B-lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

The role of the Janus-faced transcription factor PAX5-JAK2 in acute lymphoblastic leukemia

AbstractPAX5-JAK2 has recently been identified as a novel recurrent fusion gene in B-cell precursor acute lymphoblastic leukemia, but the function of the encoded chimeric protein has not yet been characterized in detail. Herein we show that the PAX5-JAK2 chimera, which consists of the DNA-binding paired domain of PAX5 and the active kinase domain of JAK2, is a nuclear protein that has the ability to bind to wild-type PAX5 target loci. Moreover, our data provide compelling evidence that PAX5-JAK2 functions as a nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream signal transducers and activators of transcription (STATs) in an apparently noncanonical mode. The chimeric protein also enables cytokine-independent growth of Ba/F3 cells and therefore possesses transforming potential. Importantly, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. Together, our data show that PAX5-JAK2 simultaneously deregulates the PAX5 downstream transcriptional program and activates the Janus kinase-STAT signaling cascade and thus, by interfering with these two important pathways, may promote leukemogenesis.

Functional Heterogeneity of PAX5 Chimeras Reveals Insight for Leukemia Development

PAX5, a transcription factor pivotal for B-cell commitment and maintenance, is one of the most frequent targets of somatic mutations in B-cell precursor acute lymphoblastic leukemia. A number of PAX5 rearrangements result in the expression of in-frame fusion genes encoding chimeric proteins, which at the N-terminus consistently retain the PAX5 DNA-binding paired domain fused to the C-terminal domains of a markedly heterogeneous group of fusion partners. PAX5 fusion proteins are thought to function as aberrant transcription factors, which antagonize wild-type PAX5 activity. To gain mechanistic insight into the role of PAX5 fusion proteins in leukemogenesis, the biochemical and functional properties of uncharacterized fusions: PAX5-DACH1, PAX5-DACH2, PAX5-ETV6, PAX5-HIPK1, and PAX5-POM121 were ascertained. Independent of the subcellular distribution of the wild-type partner proteins, ectopic expression of all PAX5 fusion proteins showed a predominant nuclear localization, and by chromatin immunoprecipitation all of the chimeric proteins exhibited binding to endogenous PAX5 target sequences. Furthermore, consistent with the presence of potential oligomerization motifs provided by the partner proteins, the self-interaction capability of several fusion proteins was confirmed. Remarkably, a subset of the PAX5 fusion proteins conferred CD79A promoter activity; however, in contrast with wild-type PAX5, the fusion proteins were unable to induce Cd79a transcription in a murine plasmacytoma cell line. These data show that leukemia-associated PAX5 fusion proteins share some dominating characteristics such as nuclear localization and DNA binding but also show distinctive features. This comparative study of multiple PAX5 fusion proteins demonstrates both common and unique properties, which likely dictate their function and impact on leukemia development.

Inherited susceptibility to pre B-ALL caused by germline transmission of PAX5 c.547G&gt;A

A single-nucleotide polymorphism (SNP) in PAX5 leading to an amino-acid change in the octapeptide domain at position c.547G>A (p.Gly183Ser) has recently been described to confer an inherited susceptibility for childhood pre B-ALL.1 This susceptibility was transmitted autosomal dominant in two independent families with variable penetrance and aberrations of chromosomal part 9p resulting in loss of the wild-type (wt) PAX5 allele occurruring simultaneously in the leukemic cells.

PAX5 Fusion Genes Activate The STAT5 Signaling Pathway Through Lck Over-Expression

PAX5 is a transcription factor acting both as an activator and a repressor, known to be frequently altered (30% of the cases) in B Cell Precursor Leukemia (BCP-ALL). PAX5 aberrations include point mutations, deletions, amplifications and translocations with several partner genes. All the originated fusion genes retain the PAX5 DNA binding domain but acquire the regulatory regions of the partner gene, showing a dominant negative mechanism on endogenous PAX5. However, the function of PAX5 fusion proteins and their common mechanisms of leukemogenesis are poorly understood.We have established an in vitro model in which wild-type murine pre-BI cells were transduced with the most recurrent PAX5 fusion gene, PAX5/ETV6. Gene expression profiling of transduced PAX5/ETV6 pre-BI cells showed a significantly altered transcription profile compared to empty vector control. Interestingly, pathway analyses revealed that many of the genes implicated in the B-cell receptor (BCR) assembly and signaling were repressed by PAX5/ETV6. In addition, the downstream signaling of the BCR was non-functional through a block in IgM heavy chain rearrangement.Aim of the current study has been to elucidate the signaling pathway activated either in PAX5/ETV6 transduced pre-BI cells and in primary cells from patients carrying different PAX5 translocations, in order to overcome the absence of a functional BCR expression.Interestingly, among the top-ranked over-expressed genes in PAX5/ETV6 transduced pre-BI cells, the PAX5 repressed target gene LCK was up-regulated both at basal level (mean fold change by RQ-PCR in 3 different pre-BI lines: 1.79, p<0.001) and at all time points (0, 24, 48 and 72 hours) after o.n. IL-7 starvation (mean FC: 8.0, range 3.8-15.9, p<0.01). LCK is a src family kinase involved in STAT5 signaling pathway and known to play an important role in T cell survival.Indeed, for the first time we found LCK as significantly up-regulated in primary samples of patients carrying different PAX5 translocations, including PAX5/AUTS2, PAX5/CHFR, PAX5/SOX5 and PAX5/POM121C, when compared to BCP-ALL cases with wild-type PAX5, as shown by microarray analyses and confirmed by RT RQ-PCR (mean fold increase: 6.3, p=0.027).Strikingly, in addition to LCK overexpression, PAX5/ETV6 induction in pre-BI cells dephosphorylates the inhibitory domain of LCK (LCK Y505) (mean difference in LCKY505/total LCK ratio: 63%, ranging from 38% to 81% in 3 different pre-BI lines), thus hyper-activating the LCK signaling. By phosphoflow analysis we also demonstrated LCK-dependent STAT5 hyper-phosphorylation after IL7 stimulation at all time points in time course experiments (mean fold change: 2.0, range 1.77-2.18). Downstream in the pathway, as a confirmation, we found by RQ-PCR the up-regulation of cMYC, a STAT5 target (FC: 2.65, p<0.01, at 30 minutes and 3.85, p<0.01, at 24h after IL-7 stimulation). Also CCND2, a cMYC target, was upregulated (FC: 2.27, p<0.001, at 30 minutes and 1.83, p<0.001, at 24h after IL-7 stimulation). Moreover, PAX5/ETV6 induced pre-BI cells show an increase in the replicative S phase cells at all time points after o.n. IL7 withdrawal (+58%, +39% and 61%, p<0.001, for PAX5/ETV6 after 0, 24 and 48 hours, respectively) and a faster proliferation rate, likely due to a different STAT5 activation profile. This mechanism is specific and reversible, as the LCK inhibitor BIBF1120 reduces STAT5 phosphorylation in PAX5/ETV6 cells (-11% and -46% after 24 and 48 hours, respectively), thus decreasing the fraction of S phase cells (-20%, p<0.01 and -31%, p<0.001, after 24 and 48 hours, respectively) and leading to a proliferation rate comparable to the control cells.These results further sustain the role of PAX5 fusion proteins as aberrant transcriptional factors, leading to the over-expression and hyper-activation of LCK, with a role in the activation of signaling pathways alternative to BCR (i.e. STAT5 hyper-phosphorylation) to sustain survival of leukemic cells. The further comprehension of the signaling mechanisms activated by PAX5 fusion genes will be fundamental for the development of new strategies to arrest tumor proliferation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation

Abstract 1109Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C). [Display omitted] Disclosures:Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

Dominant-negative mechanism of leukemogenic PAX5 fusions

PAX5 encodes a master regulator of B-cell development. It fuses to other genes associated with acute lymphoblastoid leukemia (ALL). These fusion products are potent dominant-negative (DN) inhibitors of wild-type PAX5 resulting in a blockade of B-cell differentiation. Here, we show that multimerization of PAX5 DNA-binding domain (DBD) is necessary and sufficient to cause extremely stable chromatin binding and DN-activity. ALL-associated PAX5-C20S results from fusion of the N-terminal region of PAX5 including its paired DBD, to the C-terminus of C20orf112, a protein of unknown function. We report that PAX5-C20S is a tetramer which interacts extraordinarily stably with chromatin as determined by fluorescence recovery after photobleaching (FRAP) in living cells. Tetramerization, stable chromatin-binding and DN-activity all require a putative five-turn amphipathic α-helix at the C-terminus of C20orf112, and does not require potential co-repressor binding peptides elsewhere in the sequence. In vitro, the monomeric PAX5 DBD and PAX5-C20S binds a PAX5-binding site with equal affinity when it is at the center of an oligonucleotide too short to bind to more than one PAX5 DBD. But PAX5-C20S binds the same sequence with tenfold higher affinity than the monomeric PAX5 DBD when it is in a long DNA molecule. We suggest that the increased affinity results from interactions of one or more of the additional DBDs with neighboring non-specific sites in a long DNA molecule, and that this can account for the increased stability of PAX5-C20S chromatin binding compared to wt PAX5, resulting in DN-activity by competition for binding to PAX5-target sites. Consistent with this model, the ALL-associated PAX5 fused to ETV6 or the multimerization domain of ETV6 SAM results in stable chromatin binding and DN-activity. In addition, PAX5 DBD fused to artificial dimerization, trimerization, and tetramerization domains result in parallel increases in the stability of chromatin binding and DN-activity. Our studies suggest that oncogenic fusion proteins that retain the DBD of the transcription factor and the multimerization sequence of the partner protein can act in a DN fashion by multimerizing and binding avidly to gene targets preventing the normal transcription factor from binding and inducing expression of its target genes. Inhibition of this multimeriztion may provide a novel therapeutic approach for cancers with this or similar fusion proteins.

202 The Reduced and Altered Activities of PAX5 are Linked to the Protein-Protein Interaction Motif (Coiled-Coil Domain) of the PAX5-PML Fusion Protein in t(9;15)-Associated Acute Lymphocytic Leukemia

Abstract 1022 The paired box domain of PAX5 was reported to fuse with the sequence of promylocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia (B-ALL). In the present studies, we found, by gel-shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction (RQ-PCR) analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil protein interaction domain located within PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared to wild-type PAX5. Furthermore, the dimerization domain (coiled-coil) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the coiled-coil domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins. Disclosures: No relevant conflicts of interest to declare.

Abstract 2171: The fusion protein PAX5-JAK2 constitutively activates JAK-STAT signaling

Abstract In B-cell precursor acute lymphoblastic leukemia the transcription factor PAX5, a master regulator of B-cell commitment and maintenance, is recurrently fused to several different partner proteins including other transcription factors, structural proteins, and the tyrosine kinase (TK) JAK2. Mutations and translocations affecting JAK2 are found in a variety of hematopoietic malignancies and lead to its constitutive activation and cytokine-independent JAK-STAT signaling. The chimeric PAX5-JAK2 protein contains the DNA binding domain of PAX5 and the JH1 kinase domain of JAK2. Reciprocal JAK2-PAX5 transcripts are also expressed, and the respective JAK2-PAX5 fusion protein consists of the JAK2 JH2-JH7 domains fused to the PAX5 partial homeodomain and the transactivation and inhibitory domains. In a first step towards the understanding of the function of these chimeric proteins, their subcellular localization was determined. Transfection of tagged fusion proteins into HEK293 and HeLa cells displayed nuclear localization of PAX5-JAK2. In contrast, JAK2-PAX5 fusion proteins were mainly localized in the cytoplasm. In order to determine whether PAX5-JAK2 is tyrosine phosphorylated and whether PAX5-JAK2 or JAK2-PAX5 is capable of activating STAT proteins, intracellular phosphoprotein analyses using flow cytometry were performed. Transiently transfected unstimulated JAK2-deficient human gamma2A cells exhibited a high degree of phosphorylation of PAX5-JAK2 compared to JAK2 wild-type protein, strongly suggesting cytokine stimulation-independent TK activity of the fusion protein. Additionally, expression of PAX5-JAK2 but not JAK2-PAX5 resulted in constitutive phosphorylation of STATs 1, 3, and 5. The capability of PAX5-JAK2 to induce STAT5 was confirmed by reporter gene assays. Mutation of the respective tyrosine residues in PAX5-JAK2 completely abolished the phosphorylation of STATs demonstrating that their activation is phospho-PAX5-JAK2-dependent. As it has been shown that PAX5 fusion proteins can act as competitive inhibitors of wild-type PAX5, we also performed a CD19 luciferase assay which showed that PAX5-JAK2 has a negative effect on the activation capacity of wild-type protein. Additional analysis including gene expression profiling of patient material will be performed to further evaluate the function of the chimeric proteins. Together, PAX5-JAK2 appears to affect JAK-STAT signaling and may at the same time interfere with normal B-cell development. In contrast to ETV6-JAK2 – the only other JAK2 chimeric protein analyzed in detail to date – which resides in the cytoplasm and is tyrosine phosphorylated upon dimerization, PAX5-JAK2 localizes to the nucleus and lacks any putative self-association motif. Hence, PAX5-JAK2 represents the first nuclear JAK2-fusion protein, which constitutively activates the JAK-STAT signaling pathway. This project is funded by the Austrian Science Fund FWF (P21554). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2011-2171

The Reduced and Altered Activities of PAX5 Are Linked to the Protein-Protein Interaction Motif (coiled-coil domain) of the PAX5-PML Fusion Protein In t(9;15)-Associated Acute Lymphocytic Leukemia.

AbstractAbstract 1022The paired box domain of PAX5 was reported to fuse with the sequence of promylocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia (B-ALL). In the present studies, we found, by gel-shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction (RQ-PCR) analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil protein interaction domain located within PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared to wild-type PAX5. Furthermore, the dimerization domain (coiled-coil) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the coiled-coil domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins. Disclosures:No relevant conflicts of interest to declare.

Dominant Negative Form of PAX5 Is Generated by Multimerization of Its DNA Binding Domain

AbstractAbstract 25We have previously cloned a number of fusion genes involving PAX5 in acute lymphoblastic leukemia (ALL) (Kawamata N. et al. PNAS, 2008). All of these fusion products exerted a dominant negative effect over the wild-type PAX5. One of these fusion PAX5 proteins, PAX5-C20orf112, was generated by the fusion between the DNA binding domain of PAX5 (PAX5DB) and the C-terminal end of C20orf112. To find the mechanism of the dominant negative effect of the PAX5-C20 fusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assay using PAX5-C20 and PAX5wt constructs connected with Yellow Fluorescence Proteins (YFP). Results showed extremely strong DNA binding affinity of PAX5-C20 compared to PAX5wt. FRAP experiments using deletion mutants of PAX5-C20 showed that both the DNA binding domain and C-terminal alpha-helix region of C20 were indispensable for this strong binding to DNA. Fluorescence Resonance Energy Transfer (FRET) assay, Bi-molecule Fluorescence Complementation (BiFC) assay, and co-immunoprecipitation assay showed that C-terminal end of C20 containing an alpha-helix region encodes a homo-multimerization domain. To confirm that homo-multimerization of PAX5DB increases DNA binding affinity, PAX5DB was fused to the inducible dimerization motif of FKBP (PAX5DB-FK). PAX5DB-FK increased its DNA binding affinity with addition of FKBP ligand inducing homo-dimerization. We also fused PAX5DB to homo-dimerization of MAX (bHLH domain), or tetramerization domain of TP53. FRAP assays showed that homo-dimerization increased its DNA binding activity, and homo-tetramerization further increased its DNA binding and its dominant negative effect over PAX5wt. PAX5-ETV6, also a common fusion protein in ALL, exerts a dominant negative effect over PAX5wt. The ETV6 region of this fusion protein has a multimerization (SAM) domain and the PAX5DB-ETV6SAM mutant protein also showed a dominant negative effect and strong binding to DNA. Importantly, in further studies, co-expression of PAX5-C20 and the YFP-C20-alpha-helix-region diminished the strong DNA binding and the dominant negative activity of the fusion protein. Our data show that multimerization of the DNA binding domain of PAX5 induces strong DNA binding activity, leading to its dominant negative effect over the wild type transcription factor. We believe this represents a new paradigm explaining how a number of fusion genes containing a DB motif from one protein and a multimerization motif from the other partner, can behave in a dominant negative fashion. These observations suggest that peptides/ small molecules inhibiting the multimerization of these oncogenic fusion transcription factors can be promising reagents for treating cancers. Disclosures:No relevant conflicts of interest to declare.

Dominant-Negative Impact of PAX5/TEL on Downstream Targets of PAX5 and Essential Pre-B Cell Receptor Genes

AbstractAbstract 3231B cell lineage acute lymphoblastic leukemia (ALL) is a common malignancy in childhood, and the pre-B cell receptor (pre-BCR) signalling pathway was previously demonstrated to function as a tumor suppressor. The transcription factor PAX5, a key regulator of B cell development, is frequently involved in chromosomal rearrangements of leukemic blasts. Using high resolution single nucleotide polymorphism (SNP) genomic microarray by us and other groups, several candidate partner genes fused to PAX5 have been detected in samples of pediatric ALL, such as TEL, FOXP1, AUTS2, and C20orf112. Recently, we studied the fusion gene PAX5/TEL and its role in leukemic evolution by Affymetrix HG-U133 plus 2.0 Array of the ALL cell line Nalm6 transfected with a PAX5/TEL construct. PAX5/TEL reduced the expression of PAX5 and its downstream target genes (e.g. CD79A, BACH2, CD19). Moreover, we demonstrated a dominant negative impact of the PAX5/TEL-fusion protein on the binding affinity of wild-type PAX5 to the promoter of CD79A (Iwanski et al., 2009 ASH Abstract No. 3455).To expand our findings, we analyzed the gene expression profile of pediatric ALL samples carrying PAX5/TEL (PAX5/TEL+, n=2) compared to samples with normal PAX5 (n=7) from a genomic ALL study. Samples with normal PAX5 were selected from among 95 B-ALL patients with normal PAX5 status, based on characteristics that most closely matched the two PAX5-TEL+ patients including cytogenetics and current risk stratification. Gene expression data were compiled using the Affymetrix HG-U133A Array, and a heatmap based on the Top 200 probes with the highest expression levels from both sample sets was generated (TIBCO Software Inc.). Notably, the downregulated genes included Bruton agammaglobulinemia tyrosine kinase (BTK; -2.8 fold, FDR < 0.2), an important regulator of pre-BCR signaling, Spleen tyrosine kinase (SYK, -2.3 fold, FDR < 0.2), and IGHM (-5,9 fold; FDR < 0.1), but also significantly up-regulated expression of genes involved in myeloid differentiation, namely Myeloperoxidase (MPO, +24.2 fold, FDR < 0.2), and CCAAT/enhancer binding protein alpha (CEBPA, +3.2 fold, FDR < 0.3), as well as the erythroid genes Aminolevulinate delta-dehydratase (ALAD, +12.2 fold, FDR < 0.2) and the Erythropoietin receptor (EPOR, +7.0, FDR < 0.3).Additionally, we performed a meta-analysis comparing deregulated genes detected in the Nalm6-microarray (PAX5/TEL-MIGR vs. empty vector) to the data from the human B-ALL samples (PAX5/TEL+ vs. normal PAX5). Overall, we identified a set of 35 overlapping genes (FDR < 0.3) that were deregulated in both data sets (21 downregulated, 14 up-regulated). Notably, 10 out of the 21 (47%) downregulated genes are known to be involved in B cell development and BCR signaling, some of them well-recognized as direct PAX5 target genes (e.g. CD79A, CD19, BACH2).Moreover, reporter gene assay with a luciferase reporter construct containing cDNA of the CD19 promoter with PAX5 binding sites (luc-CD19) was performed in Nalm6 cells. Since these cells already express a high level of endogenous PAX5, transcriptional activity of the luc-CD19 reporter plasmid was relatively high in the Nalm6 cells transfected with empty vector, as compared to 293T cells. In contrast, PAX5/TEL-transduced Nalm6 cells displayed a significantly reduced transcriptional activation of the reporter construct (P < 0.01).We also explored if mutation and/or deletion of PAX5 (mut/del PAX5) may have an impact on genes involved in B cell development and the pre-BCR/BCR pathway. Hence, expression files from human ALL samples with mut/del PAX5 (n=50) and B-ALL samples with normal PAX5 (n=95) were analyzed. Notably, only two genes that are known to be involved in B cell development and the pre-BCR pathway were significantly downregulated in samples with mut/del PAX5 compared to normal PAX5, namely CD72, a B cell specific repressor gene activated by PAX5 (-1.51 mean fold, FDR = 0.05), and immunoglobulin heavy constant delta (IGHD), a gene involved in pre-BCR signalling (-1.6 mean fold, FDR = 0.18). These findings suggest no strong influence of mut/del PAX5 on the expression of downstream genes involved in pre-BCR signaling.In conclusion, our results provide further insights into the dominant-negative role of PAX5/TEL and link this fusion gene with the pre-BCR pathway. Disclosures:No relevant conflicts of interest to declare.

The reduced and altered activities of PAX5 are linked to the protein–protein interaction motif (coiled-coil domain) of the PAX5–PML fusion protein in t(9;15)-associated acute lymphocytic leukemia

The paired box domain of PAX5 was reported to fuse with the sequence of promyelocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia. In the present studies, we found, by gel shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence acts as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil (CC) protein interaction domain located within the PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but not PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared with wild-type PAX5. Furthermore, the dimerization domain (CC) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the CC domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins.

The Pax5 Fusion Product Pax5-C20orf112 Causes Downregulation of Pre-B Cell Receptor Genes and Induces Differential Proliferation Patterns in B-Lymphoblastic Cell Lines.

Abstract 1284Poster Board I-306Recent SNP array analyses of B-acute lymphoblastic leukemia (B-ALL) have identified disruptions of the gene encoding the B-cell specific transcription factor Pax5 as one of the most common genomic lesions in this disease (> 30%); it being hemizygously deleted, mutated or involved in translocations. Pax5 translocates and forms fusion products with at least 12 different partners including C20orf112, leading to a chimeric Pax5/C20orf112 (Pax5/C20s) protein. Pax5 fusion products act as dominant negatives, competing for promoter binding sites with wild type (wt) Pax5 and thereby deregulating expression of target genes. In order to elucidate the molecular effects of fusion products involving the Pax5 gene, we performed a global gene expression analysis in the Nalm-6 B-ALL cell line. The cells were transfected with MSCV expression plasmids containing either empty vector, wild type Pax5 or a short fusion product of Pax5 and C20orf112 (Pax5/C20s), each containing IRES sequences for co-expression of GFP. Overexpression of Pax5 and Pax5/C20s was confirmed by western blot and quantitative RT PCR. RNA was extracted from cells sorted by FACS for GFP and processed for hybridization on Affymetrix HG-U133 plus 2 gene expression microarrays. Candidate genes were validated with RT real time PCR. Among the most differentially downregulated genes by the Pax5/C20s fusion product were candidate genes such as pleckstrin homology domain containing, family A member 2 (PLEKHA2) (12.64-fold), B-cell associated transcription factors POU class 2 associating factor 1 (POU2AF1) (4.4-fold) and transcription factor 3 (TCF3, E2A) (3.9-fold). Another intriguing observation was the downregulation of a group of genes associated with signaling through the pre-B cell receptor such as phosphoinositide-3-kinase adaptor protein 1 (BCAP) (3.35 fold), immunoglobulin heavy locus (IGH) (2.8 fold), pre-B lymphocyte genes -3 and -1 (VPREB3, VPREB1) (2.6-fold and 1.75-fold, respectively), spleen tyrosine kinase (SYK) (1.6 fold) and B-cell linker (SLP65, BLNK) (1.5-fold) by the Pax5/C20s fusion product. For stable expression and growth curves, Nalm6, 697, Kasumi2, RCH-ACV, SEM, HPB-Null, BV173 and BEL1 B-lymphoblastic cell lines were infected with retroviruses expressing the above mentioned retroviral expression constructs. We noted that forced expression of the PAX5/C20s fusion product inhibited growth in cell lines, which had functional pre-B cell receptor signaling. In contrast, the fusion gene either did not affect or enhanced growth of B-ALL cell lines, in which expression of a functional pre-B cell receptor was missing. Of note, Pax5 wt caused growth inhibition in B-ALL cell lines lacking functional pre-B cell receptor signaling. In cells with functional pre-B cell signaling, the response to engagement of the receptor as measured by calcium flux assay was diminished by overexpression of the Pax5/C20s fusion product as compared to empty vector control or PAX5 wt. These results suggest that the mechanisms of leukemogenesis of Pax5/C20s in ALL cells may be dependent on the functionality of the pre-B cell receptor pathway. This could be of great therapeutic value as it would potentially allow ALL cells to be divided into two different subtypes depending on pre-B cell receptor functionality and possibly identify the pre-B cell receptor pathway as a new therapeutic target. DisclosuresNo relevant conflicts of interest to declare.