VpreB Surrogate Light Chain Expression in B-Lineage ALL: A Report from the Children's Oncology Group

Immunotherapies directed against B-cell surface markers have been a common developmental strategy to treat B-cell malignancies. The IgH surrogate light chain (SLC), comprised of the VpreB1 (CD179a) and Lamda5 (CD179b) subunits is expressed on pro- and pre-B cells where it governs preBCR-mediated autonomous survival signaling. We hypothesized that the pre-BCR might merit the development of targeted immunotherapies to decouple "autonomous" signaling in B-lineage acute lymphoblastic leukemia (B-ALL). We used the COG minimal residual disease (MRD) flow panel to assess pre-BCR expression in 36 primary patient samples accrued to COG standard and high-risk B-ALL studies through AALL03B1. We also assessed CD179a expression in 16 cases with Day 29 end-induction samples, pre-selected to have ≥1% MRD. All analyses were performed on a 6-color Becton-Dickinson flow cytometer in a CLIA/CAP-certified laboratory. Among 36 cases tested, thirty-two were at the pre-B and four were at the pro-B stages of developmental arrest. One or both mAbs showed that CD179a was present in ≥20% of the B-lymphoblast population. All cases expressed CD179a in the end-induction B-lymphoblast population. The CD179a component of the SLC is commonly expressed in B-ALL, regardless of genotype, stage of developmental arrest or NCI risk-status.

Pre-B cell receptor expression in B-lineage acute lymphoblastic leukemia: A report from the Children’s Oncology Group.

e19006 Background: The surface expression of mature B-cell markers have led to the development of immunotherapies against B-lineage lymphoblastic leukemia/lymphoma (B-ALL/B-LLy). Relapsing clones that have altered surface antigen expression are common means of treatment failure with immunotherapies. The elimination of the pan-B cell repertoire by current B-cell immunotherapies contributes to immune-compromise. A promising target is the pre-BCR surrogate light chain, comprised of the VpreB1 (CD179a) and Lamda5 (CD179b) subunits. Surrogate light chain is expressed on pro- and pre-B cells where it governs preBCR-mediated autonomous survival during B-cell maturation. Gene expression analyses have shown that CD179a is expressed in a sub-set of 10 to 15% of B-ALL cases. Because immunotherapies targeted to restricted stages of B-cell development may overcome the limitations of pan B-cell ablation, we tested the hypothesis that CD179a is more commonly expressed on B-lymphoblasts than previously thought. Methods: Utilizing an annotated set of 36 standard (AALL0331) and high-risk (AALL0232) B-ALL cases accrued to Children’s Oncology Group AALL03B1, we adapted the COG minimal residual disease (MRD) flow panel to include two additional PE- and FITC-conjugated mAbs against CD179a (Biolegend and i2Pharma). We assessed CD179a expression in 16 cases for which we had Day 28 end-induction samples, pre-selected to have ≥1% MRD, as determined by the COG Reference laboratories. Cases with ≥20% CD179a surface expression were determined to be positive for statistical comparisons. All analyses were performed on a 6-color Becton-Dickinson flow cytometer in a CLIA/CAP certified laboratory. Results: Thirty-four cases were arrested at the CD10-positive pre-B stage, and two cases at the CD10-negative pro-B stage. One or both mAbs showed that CD179a was present in ≥20% of the B-lymphoblast population, ranging from 20.2% to 90.6% for all 36 diagnostic samples. All cases expressed CD179a in the end-induction B-lymphoblast population. Compared to gene-expression based predictions, we found a significant difference between expected versus observed flow-based CD179a positivity (two-sided Fisher’s exact test, P< 0.001). We found that CD179a expression was observed in cases having E2A-PBX3, KMT2A, BCR-ABL1 and other re-arrangements that typify mixed phenotype acute leukemias (MPALs). Conclusions: Our results show that CD179a is commonly expressed in B-ALL, regardless of stage, NCI risk features, or molecular aberrations. Because the productively assembled preBCR mediates autonomous survival signaling in pro- and pre-B cells, it may also contribute to the mechanistic basis of MRD in B-ALL. Immunotherapies directed against the CD179a component of the preBCR may spare the immune-compromise that occurs with pan B-cell ablation, and prevent the emergence of therapy-resistant disease in B-ALL/B-LLy.

A Germline-Encoded Structural Arginine Trap Underlies the Anti-DNA Reactivity of a Murine V Gene Segment

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

CRISPR/Cas9 mediated knock-out of VPREB1 gene induces a cytotoxic effect in myeloma cells

Background Multiple Myeloma (MM) is a heterogeneous, hematological neoplasm that accounts 2% of all cancers. Although, autologous stem cell transplantation and chemotherapy are currently the most effective therapy, it carries a notable hazards, in addition for being non curative. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has been successfully tried at the experimental level, for the treatment of several hematological malignancies. Objectives We aimed to investigate the in-vitro effect of CRISPR-cas9-mediated knock-out of V-set pre B-cell surrogate light chain 1”VPREB1” gene on the malignant proliferation of primary cultured myeloma cells. Methods Bioinformatics’ analysis was performed to explore the gene expression profile of MM, and the VPREB1 gene was selected as a target gene for this study. We knocked-out the VPREB1 gene in primary cultured myeloma cells using CRISPR-cas9, the VPREB1 gene editing efficacy was verified by determining VPREB1 gene expression at both the mRNA and protein levels by qPCR and immunofluorescence, respectively. Furthermore, the cytotoxic effect on primary myeloma cells proliferation was evaluated using cytotoxicity assay. Results There was a statistically significant reduction of both VPREB1 mRNA and protein expression levels (p<0.01). knock-out of VPREB1 gene in myeloma cell line resulted in a statistically significant reduction of myeloma cell proliferation. Conclusion CRISPR-cas9-mediated knock-out of VPREB1 gene is effective for inhibiting the proliferation of primary myeloma cells. This would provide a basis for a promising therapeutic strategy for patients with multiple myeloma.

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This ‘alternate pathway’ of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Pre-BCR Surrogate Light Chain Components VPREB1 and IGLL1 Function As Pre-BCR-Independent Tumor Suppressors in Acute Lymphoblastic Leukemia

Background: IGLL1 and VPREB1 encode for the l5 and VpreB components, respectively, of the surrogate light chain (SLC) of the pre-B cell receptor (pre-BCR). During early B-cell development, immunoglobulin (Ig) heavy chains pairs with SLCs to form the pre-BCR, a central signaling unit that drives proliferation and survival. Accordingly, germline mutations of IGLL1 (Minegishi J Exp Med 1998) and VPREB1 (Conley Immunol Rev 2005) are associated with profound B-cell defects and agammaglobulinemia in humans. However, somatic deletions of VPREB1 gene are frequent lesions in B-ALL and occur in >10% of B-ALL cases (Mangum et al., Leukemia 2014; Geng et al., Cancer Cell 2015), the significance of which is not clear. Since VPREB1 deletions are typically present at the time of diagnosis and are rarely acquired as secondary lesions at the time of relapse (Kuster Blood 2011), we hypothesized that loss of VPREB1 represents an early and essential event in leukemogenesis. Experimental Approach and Results: For genetic gain and loss of function analysis of VPREB1 and IGLL1, we established leukemia models based on pre-B cells from Igll1-/- mice (Kitamura Cell 1992), and Vpreb1-Igll1 double-transgenic mice (Van Loo Immunity 2007). Loss of Igll1 completely abrogated SLC expression on the surface of pre-B cells. In contrast, VpreB1-Igll1 double-transgenic pre-B cells expressed constitutively higher surface levels of SLC as part of their pre-BCR as evidenced by flow cytometry. Compared to wildtype controls, Igll1-/- pre-B cells lacking the ability to express a functional SLCs were more readily transformed by BCR-ABL1 oncogene. However, pre-B cells of VpreB1-Igll1 transgenic mice, were not permissive to BCR-ABL1 mediated transformation. In agreement with these results, VpreB1-Igll1 double-transgenic pre-B cells were resistant transformation by BCR-ABL1 in vivo. BCR-ABL1-transgenic mice with enforced expression Vpreb1-Igll1 remained disease-free for more than nine months, whereas the vast majority of BCR-ABL1-transgenic mice downregulated pre-BCR surface expression and developed lethal B-ALL within 90 days of birth (n=34, P<0.0001). Compared to wildtype pre-B cells, double-transgenic expression of VpreB-Igll1 interfered with oncogenic BCR-ABL1 tyrosine kinase signaling and suppressed phosphorylation of Btk, Syk and Src kinases resulting in cell cycle arrest and reduced colony formation ability. To test whether SLC tumor suppressive function depends on pre-BCR activity, we studied BCR-ABL1-mediated transformation of VpreB1-Igll1 double-transgenic pre-B cells on a Rag1-deficient background. Rag1-/- pro-B cells lack the ability to rearrange Ig V, D and J-gene segments and cannot express a functional Ig mHC, the central structural element of the pre-BCR. Surprisingly, Rag1-/-VpreB1-Igll1 double-transgenic pro-B cells were also resistant to BCR-ABL1-mediated transformation. These findings provide genetic evidence that Vpreb1 and Igll1 exert tumor suppressive effect on B cells regardless of functional pre-BCR expression. Conclusion: The VpreB and Igll1 surrogate light chain components of the pre-BCR act as a tumor suppressors in pre-B ALL cells. Interestingly, the tumor suppressor function of both VpreB and Igll1 is independent from their SLC-role in pre-BCR signaling: Vpreb1- and Iggl1-mediated tumor suppression was effective, regardless of pre-BCR function. These findings provide genetic evidence that VpreB and Igll1 have pre-BCR- and SLC-independent functions that prevent malignant transformation and limit proliferation of normal pre-B cells. Our findings support the hypothesis that VPREB1 deletion represents an early event during clonal evolution towards pre-B ALL, facilitating subsequent steps of leukemic transformation. Disclosures No relevant conflicts of interest to declare.

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

SUMMARYB-1a cells are long-lived, self-renewing innate like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells they have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver (FL) versus bone marrow (BM) environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa (Igk) recombination at the early pro-B cell stage. As a result, B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain (SLC). This ‘alternate pathway’ of development enables the production of B cells with self reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing models of B-1a cell development and explain how these cells acquire their unique properties.

Analysis of 54 Follicular Lymphomas By Whole Exome Sequencing Identifies Multiple Novel Recurrently Mutated Pathways

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving. Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing. Selected mutations were validated in an expansion cohort of 120 FL. Results: We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119). RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB. To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC. We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting. Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells. We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis. We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor. Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples. Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL. Disclosures Malek: Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.