IKZF1 Mutations Research Articles

A Point Mutation in IKAROS ZF1 Causes a B Cell Deficiency in Mice.

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N-ethyl-N-nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1L132P Furthermore, Ikzf1+/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1L132P/L132P mice.

Analysis of Clinical Characteristics and Prognosis in Children with Acute Megakaryoblastic Leukemia without Down Syndrome

To analyze the clinical characteristics and treatment effects of children with acute megakaryoblastic leukemia without down syndrome (non-DS-AMKL). The clinical data of 19 children with non-DS-AMKL treated in the Pediatric Hematology Ward in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from May 2008 to April 2018 were analyzed retrospectively. The clinical characteristics, laboratory test and treatment methods of the children were concluded. All patients were followed up to evaluate the effect of treatment. The 19 cases of children included nine male and ten female, the median age of onset was 2 years old. The clinical manifestations showed nonspecific. The median white blood cell of peripheral blood was 15.88×109/L, the median hemoglobin was 67 g/L and median platelet was 16×109/L. An increase of primitive and naive megakaryocytes was found in the bone marrow sample. The immunophenotypes of bone marrow detected by flow cytometry in 19 children were all positive expressed for CD41, CD61. Genetic tests showed that five cases carrying EVI1, including one complicating with MLL/AF10, one patient carrying HOX11, one carrying GATA1, IKZF1 and DDX11 mutations, one patient carrying missense mutation of NRAS, one with missense mutation of KRAS and two cases with high expression of WT1. Karyotype analysis were performed in 11 cases of children, including four with normal karyotype, four with complex karyotypes, one with trisomy 8, one with 7/13 trisomy 21 without Down syndrome manifestations (considered as abnormal somatic karyotype) and one Robertsonian translocation carrier involving chromosomes 14 and 21. Ten children received treatment, including three cases with allogeneic hematopoietic stem cell transplantation (allo-HSCT) after complete remission (CR), two cases with complete donor implantation, and one without implanted but hematopoietic recovered, these three children were followed up for 26, 15 and 12 months respectively and the minimal residual disease (MRD) were all less than 10-4. Another three cases achieving CR after chemotherapy but relapsed in 5, 10 and 12 months after the onset respectively, and all the three children were died eventually. One children died of pulmonary hemorrhage during chemotherapy and three children died from discontinuation of treatment after non remission. The remaining nine children died because of without chemotherapy treatment. Non-DS-AMKL was rare in children and difficult to be diagnosed. Determination of MICM classification as early as possible was helpful for diagnosis, and genetic testing played an important role for diagnosis and prognosis evaluation. Early hematopoietic stem cell transplantation in patients with CR after chemotherapy might be an effective way to cure AMKL.

Genomic characterization of relapsed acute myeloid leukemia reveals novel putative therapeutic targets

AbstractRelapse is the leading cause of death of adult and pediatric patients with acute myeloid leukemia (AML). Numerous studies have helped to elucidate the complex mutational landscape at diagnosis of AML, leading to improved risk stratification and new therapeutic options. However, multi–whole-genome studies of adult and pediatric AML at relapse are necessary for further advances. To this end, we performed whole-genome and whole-exome sequencing analyses of longitudinal diagnosis, relapse, and/or primary resistant specimens from 48 adult and 25 pediatric patients with AML. We identified mutations recurrently gained at relapse in ARID1A and CSF1R, both of which represent potentially actionable therapeutic alternatives. Further, we report specific differences in the mutational spectrum between adult vs pediatric relapsed AML, with MGA and H3F3A p.Lys28Met mutations recurrently found at relapse in adults, whereas internal tandem duplications in UBTF were identified solely in children. Finally, our study revealed recurrent mutations in IKZF1, KANSL1, and NIPBL at relapse. All of the mentioned genes have either never been reported at diagnosis in de novo AML or have been reported at low frequency, suggesting important roles for these alterations predominantly in disease progression and/or resistance to therapy. Our findings shed further light on the complexity of relapsed AML and identified previously unappreciated alterations that may lead to improved outcomes through personalized medicine.

IKAROS-Associated Diseases in 2020: Genotypes, Phenotypes, and Outcomes in Primary Immune Deficiency/Inborn Errors of Immunity

IKAROS, encoded by IKZF1, is a zinc finger transcription factor and a critical regulator of hematopoiesis. Mutations in IKZF1 have been implicated in immune deficiency, autoimmunity, and malignancy in humans. Somatic IKZF1 loss-of-function mutations and deletions have been shown to increase predisposition to the development of B cell acute lymphoblastic leukemia (B-ALL) and associated with poor prognosis. In the last 4years, germline heterozygous IKZF1 mutations have been reported in primary immune deficiency/inborn errors of immunity. These allelic variants, acting by either haploinsufficiency or dominant negative mechanisms affecting particular functions of IKAROS, are associated with common variable immunodeficiency, combined immunodeficiency, or primarily hematologic phenotypes in affected patients. In this review, we provide an overview of genetic, clinical, and immunological manifestations in patients with IKZF1 mutations, and the molecular and cellular mechanisms that contribute to their disease as a consequence of IKAROS dysfunction.

Allogeneic HCT for adults with B-cell precursor acute lymphoblastic leukemia harboring IKZF1 gene mutations. A study by the Acute Leukemia Working Party of the EBMT.

The presence of IKZF1 gene mutations is associated with poor prognosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The goal of this retrospective study was to evaluate outcome of allogeneic hematopoietic cell transplantation (allo-HCT) in this population. Ninety-five patients transplanted in first (n = 75) or second (n = 20) complete remission (CR) from either HLA-matched sibling (n = 32), unrelated (n = 47) or haploidentical (n = 16) donor were included in the analysis. The probabilities of the overall survival (OS) and leukemia-free survival (LFS) at 2 years were 55% and 47%, respectively. Relapse incidence (RI) was 32% while non-relapse mortality (NRM), 21%. The incidence of grade II-IV acute graft-versus-host disease (GVHD) and chronic GVHD was 34% and 30%, respectively. The probability of GVHD and relapse-free survival (GRFS) was 35%. In a multivariate analysis positive minimal residual disease (MRD) status was associated with decreased chance of LFS (HR = 3.15, p = 0.004) and OS (HR = 2.37, p = 0.049) as well as increased risk of relapse (HR = 5.87, p = 0.003). Disease stage (CR2 vs. CR1) affected all, LFS, OS, GRFS, RI, and NRM. Results of allo-HCT for patients with BCP-ALL and IKZF1 mutations are generally improving, however, individuals with detectable MRD have poor prognosis and require additional intervention prior to transplantation.

Artificial thymic organoids reveal an early block in T cell differentiation in patients carrying mutations in IKZF1

Abstract IKZF1 is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. Heterozygous germline mutations in IKZF1 give rise to distinct clinical phenotypes. IKZF1 haploinsufficiency causes common variable immunodeficiency (CVID) associated with B cell immune deficiency, B-ALL susceptibility and autoimmune manifestations, without clinical T cell defects. Patients carrying dominant negative (DN) IKZF1 mutations have combined immunodeficiency (CID), with an increased proportion of naïve T cells and impaired T cell function. Mouse models of IKZF1 mutations have a distinct phenotype from patients and some models present a more severe defect in T cell development. To address these controversies and gain novel insights into the effects of distinct IKZF1 mutations on human T cell development, we used the artificial thymic organoid (ATO) system, based on the 3D aggregation and culture of a delta-like Notch ligand 4-expressing stromal cell line (MS5-Dll4) with CD34+ cells, to analyze T cell development from CD34+ cells obtained from one patient with IKZF1 haploinsufficiency and one patient with DN IKZF1 mutation. Both patients showed a similar early block in T-cell differentiation at pre-T cell stage. However, the patient with IKZF1 haploinsufficiency showed a more pronounced leakiness, with a residual production of CD3+TCRαβ+ cells, which could account for the milder T-cell phenotype. Interestingly, the DN patient presented an increased accumulation of CD4−CD8β− CD8αα+ cells. These results show an unexpected role for IKZF1 in humans in early stages of T-cell differentiation and indicate IKZF1 as a necessary factor for the induction of CD8β expression in T cells.

Ikaros regulates gene expression programs required for proper BCR-activation in mature B cells

Abstract The zinc-finger (ZnF) transcription factor Ikaros (encoded by Ikzf1) plays an essential role in regulating transcriptional programs required for B cell development, as Ikzf1null/null mice do not develop B cells. Ikaros contains two ZnF domains which function in DNA-binding and protein-protein interactions, respectively. Recently, mutations within the central DNA-binding ZnF domain of the IKZF1 gene has been linked to autoimmunity. Likewise, our murine model containing a germ-line deletion of the fourth ZnF (Ikzf1DF4/DF4) displays a B cell hyper-reactive (HR) phenotype, with B cells activated by anti-IgM without the requirement for a second co-stimulatory signal. Although Ikaros has been well-studied in early developing B cells, how loss-of-function mutations in Ikzf1 impacts downstream effector functions of mature B cells, remains largely unknown. Here, we investigated the role of Ikaros in regulating an epigenetic program required for co-stimulation-restricted activation of B cells. We utilized transcriptomic and epigenomic approaches (RNA-seq and ATAC-seq) to define transcriptional programs which dynamically change upon ex vivo stimulation and investigated their Ikaros-dependency. Interestingly, we found that the largest subset of genes deregulated between wt and Ikzf1DF4/DF4 B cells, occur prior to stimulation. In addition, CUT&RUN performed in wt B cells shows Ikaros binding to a large subset of genes expressed upon stimulation with anti-IgM and CD40L. Together, these results demonstrate that Ikaros regulates the transcriptional landscape of BCR-activated mature B cells. In the future, we will be integrating single cell RNA-seq analysis to investigate B cell heterogeneity in wt and Ikzf1DF4/DF4 B cells.

Ikaros Family Zinc Finger 1 Mutation Is an Adverse Prognostic Factor for Patients with Adult B-Cell Acute Lymphoblastic Leukemia

To investigate the prognosis of patients with adult B-cell acute lymphoblastic leukemia (B-ALL) accompanied with Ikaros family zinc finger 1 (IKZF1) mutation, and to explore the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in improving the clinical outcome of patients. The clinical data of 164 adult B-ALL patients who received IKZF-1 mutation detection by capillary electrophoresis of bone marrow were collected, and the relationship between the IKZF-1 gene mutation and the prognosis of adult B-ALL patients was analyzed. Among 164 adult B-ALL patients, the patients with IKZF-1 mutation (IKZF-1+) were 80 cases, while the patients without IKZF-1 mutation (IKZF-1-) were 84 cases. Among 80 IKZF-1 positive patients, according to the treatment method after complete remission these patients were divided into HSCT group (48 cases) and chemotherapy group (32 cases). Analysis showed that the 3-year overall survival (OS) and leukemia-free survival (LFS) rates in the IKZF1+ group were much lower. The OS and LFS rate in transplantation group were 50.3%±8.3%, 41.6%±8.5%; the OS and LFS rates in the chemotherapy group were 33.7%±12.8%, 31.5%±9.5%, which were lower than those in the IKZF1- group (the transplantation group: 79.5%±7.6%, 64.0%±8.4%; the chemotherapy group: 54.4%±9.9%, 40.6%±9.6% respectirely (P＜0.05). Among 80 B-ALL patients with IKZF-1- mutation, the 3-year OS and LFS rates were significantly higher in the transplantation group (55.3%±7.5%, 48.3%±7.6%) than those in the chemotherapy group (32.9%±11.8%, 28.4%±10.3%) with IKZF1 mutation (P＜0.05). IKZF1 mutation is an adverse prognostic factor for adult B-ALL patients, However, allo-HSCT significantly improves the OS and LFS of patients and also their clinical outcomes.

Case Report of acute myeloid leukemia with “WT1, ATRX, CEBPA, CSMD1, IKZF1, and LRP1B mutation and translocation between chromosome 1 and 19” developing from Philadelphia-negative chronic myeloid leukemia after TKI therapy

Rationale:The success of tyrosine kinase inhibitor (TKI) therapy has greatly prolonged the survival time of patients with chronic myeloid leukemia (CML), harboring the characteristic Philadelphia (Ph) chromosome. However, a fraction of patients, achieving complete cytogenetic response after TKI therapy, develop a myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with additional clonal chromosomal abnormalities in Philadelphia-negative cells (CCA/Ph–).Patient concerns:A 56-year-old woman with AML, developing from Philadelphia-negative CML after TKI therapy. She showed 6 kinds of somatic variants—CEBPA, ATRX, WT1, CSMD1, IKZF1, and LRP1B mutation after diagnosed as AML.Diagnosis:The patient was diagnosed with chronic phase CML that developed to AML after achieving durable complete cytogenetic response (CCR) and major molecular response (MMR).Interventions:The patient was treated with TKI therapy at the period of CML. When diagnosed with AML, she received induction chemotherapy regimens, consolidation therapy, and allogeneic hematopoietic stem cell transplantation subsequently.Outcomes:The patient has been CCR and MMR for nearly 4 years, and has achieved complete remission after intervention related to AML. She is now preparing for allogeneic hematopoietic stem cell transplantation.Lessons:These rare occurrences highlight the importance of exploring the relevant pathogenesis of AML developing from CML after TKI therapy. In addition to monitoring molecular changes in the course of CML, cytogenetic analysis, or next-generation sequencing of CML patients should be performed.

Clonal Competition Models to Understand Progression and Resistance in Myeloma

Background: Progression and relapse in Multiple Myeloma (MM) is induced by changes in the clonal tumor composition. In order to better understand the mechanisms underlying these dynamics, we developed clonal competition models based on the co-culture of fluorescent labelled isogenic MM cells, with or without the alteration under study. Methods: To understand the effect of mono- and bi-allelic TP53 lesions, we use the AMO1 cell line, one of few myeloma cell lines harbouring wild type TP53 (WT). After modification with CRISPR / CAS9, we selected subclones with mono- and/or bi-allelic deletion of TP53. For the characterization of alterations in RAS, we selected OPM2 cells, one of the few lines with the RAS pathway intact. Furthermore, we generated the KRAS WT, G12A and A146T sublines by stable transfection with Sleeping Beauty vectors. To study mutations related to resistance to IMIDs and PIs, we introduced mutations in the target genes IKZF1 (WT, A152T, Q170D or R439H), CUL4B (KO), and PSMB5 (WT or A20T ) in AMO1 and L363, cell lines sensitive to IMiD or PI treatment. In addition, all WT and mutant sublines were also stably transformed with E-GFP or LSS-mkate2-RFP for flow cytometry analysis. Results: We recently demonstrated that lesions in TP53, both mono- and bi-allelic, induce a growth advantage to the affected cells. In the current study, we also observed an increased fitness in KRAS mutated cells (G12A or A146T vs WT) independent of treatment. We co-cultivated KRAS mutant with WT cells at a ratio 1:3 in two independent experiments, with the color labelling switched (red/green wt/mutant and vice-versa). KRAS G12A clone significantly expanded and reached 50% of the cells at day 40. Likewise, A146T clone outcompeted WT cells, but the time required to represent the majority of cells in the coculture was longer. We next explored the effects of resistance mutations and drug exposure. Both the IKZF1 A152T and CUL4B KO mutants outcompete WT cells in the presence of Lenalidomide (LEN). The same effect was observed for the PSMB5 A20T mutant exposed to Bortezomib (BOR). This selection ("Survival Fitness") did not occur without the presence of the drug. Thus, resistance related mutations seem only to provide a fitness advantage under drug exposition. In addition, both the CUL4B KO and PSMB5 A20T mutants were overcome by WT cells when the drug was removed from co-culture, suggesting that these lesions provide a survival disadvantage without the selective pressure of IMiD or PI. This may provide an explanation for the low mutation rate in this gene in recent sequencing publications, as usually samples are not obtained under selective pressure but in treatment free intervals. IKZF1 mutations outside the IMiDs / CRBN binding area (Q170D and R439H) provided no advantage to the cells. Conclusions: Our clonal competition assays provide novel insights on the impact of point mutations on the fitness of affected myeloma subclones, either with or without the selective pressure of therapy. Figure Disclosures Martinez-Lopez: Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; F. Hoffmann-La Roche Ltd: Honoraria; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; BMS: Honoraria, Other: Advisory boards; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria.

M230 A CASE OF IKAROS FAMILY ZINC FINGER 1 (IKZF1) MUTATION CAUSING COMMON VARIABLE IMMUNODEFICIENCY

Common variable immunodeficiency (CVID) is characterized by defective antibody production leading to clinical manifestations of recurrent infections, autoimmunity/inflammatory conditions, rarely malignancy. Among the genetic mutations that lead to the CVID syndrome, heterozygous autosomal dominant mutations in gene IKZF1, encoding the transcription factor IKAROS, a member of a family of hematopoietic zinc-finger transcription factors important for lymphopoiesis, were recently identified.

Nbn−Mre11 interaction is required for tumor suppression and genomic integrity

We derived a mouse model in which a mutant form of Nbn/Nbs1mid8 (hereafter Nbnmid8) exhibits severely impaired binding to the Mre11-Rad50 core of the Mre11 complex. The Nbnmid8 allele was expressed exclusively in hematopoietic lineages (in Nbn-/mid8vav mice). Unlike Nbnflox/floxvav mice with Nbn deficiency in the bone marrow, Nbn-/mid8vav mice were viable. Nbn-/mid8vav mice hematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage-specific blockage of B cell development. Within 6 mo, Nbn-/mid8 mice developed highly penetrant T cell leukemias. Nbn-/mid8vav leukemias recapitulated mutational features of human T cell acute lymphoblastic leukemia (T-ALL), containing mutations in NOTCH1, TP53, BCL6, BCOR, and IKZF1, suggesting that Nbnmid8 mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis of Nbn-/mid8vav malignancies showed focal amplification of 9qA2, causing overexpression of MRE11 and CHK1 We propose that overexpression of MRE11 compensates for the metastable Mre11-Nbnmid8 interaction, and that selective pressure for overexpression reflects the essential role of Nbn in promoting assembly and activity of the Mre11 complex.

PS1357 FITNESS SIGNATURES IN MULTIPLE MYELOMA PROGRESSION AND RESISTANCE

Background:The amount of descriptive genetic data in multiple myeloma (MM) is growing exponentially. Clonal dynamics leading to disease progression and resistance development are evident and have been described in a number of exemplary MM patients. Still, for most identified genetic lesions, the specific impact on clinic and biology remains unclear.Aims:Aiming for a better understanding of the underlying mechanisms of clonal progression, we developed clonal competition models, based on co‐culture of fluorescence‐marked isogenic MM cells with or without the alteration under study. This experimental setting allows us to study the implications of a specific lesion and its interactions with different environmental conditions such as drug exposition.Methods:To study the effect of mono‐ and bi‐allelic TP53 lesions and the impact of proteasome subunit mutations we selected the TP53 wild‐type (WT) cell line AMO1. As this cell line has a weak response to IMiDs, we generated L363 sublines with IKZF1 or PSMB5 alterations and MM1S cells with IKZF1 or TP53 lesions. All these sublines and their parental (WT) controls were then stably marked with EGFP or LSS mKate RFP. Then, the co‐cultures with mutant and WT cells were monitored by flow cytometry under different therapeutic conditions.Results:Exploring the CoMMpass dataset, we detected one patient (MMRF_1152) who acquired two sub‐clonal nonsense CUL4B mutations after IMiD exposition. In our clonal competition assays, CUL4B KO cells were selected in the presence of LEN but they induced a survival disadvantage when the drug was not present (Figure, top). We previously described a similar observation regarding PSMB5 mutations affecting the chymotrypsin‐like catalytic core (PI binding). In vitro these alterations generated an impasse on the proteasome activity that potentially affects cell growth (Barrio S, et al. Leukemia 2019). Our results suggest that some of the acquired mutations may induce survival fitness under drug exposition but represent a disadvantage when the therapy is removed. This could explain why such alterations are not being detected more frequently in relapsed MM patients. Of note, however, a hotspot mutation identified in the LEN binding area of IKZF1 (A152T) did not have any negative impact on cell growth without treatment but was selected in the presence of LEN. IKZF1 mutations outside the LEN binding area (Q170D and R439H) did not provide any advantage to the cells. Besides survival fitness, we have also observed growth fitness, a selection that occurs independently of therapy. When cells with mono‐ or bi‐allelic TP53 lesions were co‐cultured with WT cells, a strong growth dynamic was observed. This advantage remained under exposure to Melphalan (MEL) or Bortezomib. Of interest, MEL seems to increase the fitness of cells with mono‐allelic TP53 alterations.Summary/Conclusion:In summary, we observed three different types of fitness advantages (Figure, bottom): Negative survival fitness, induced by drug exposition but with negative impact on basal cell growth as described for CUL4B or PSMB5. Neutral survival fitness (IKZF1), without this negative effect when the drug is absent. Growth fitness, independent of treatment exposition (TP53 mono‐ and bi‐allelic lesions). Altogether, our data suggest that it is possible to apply clonal competition assays to perform analytical or even quantitative genetics. This approach might help in the future to select the best regimen to control the fitness advantages induced in MM cells by patient‐specific lesions.image

PS1358 POTENT ANTI‐MYELOMA EFFICACY OF DENDRITIC CELL THERAPY IN COMBINATION WITH POMALIDOMIDE AND PROGRAMMED DEATH‐LIGAND 1 BLOCKADE IN A PRECLINICAL MODEL OF MULTIPLE MYELOMA

Background:Dendritic cell (DC)‐based vaccines are recognized as a promising immunotherapeutic strategy against cancer, and various combination approaches have been developed to enhance DC function by modulating immune responses and tumor microenvironment.Aims:In this study, we investigated the efficacy of DC vaccination in combination with pomalidomide and programmed death ligand‐1 (PD‐L1) blockade in a murine model of multiple myeloma (MM).Methods:MOPC‐315 cell lines were injected subcutaneously to establish MM bearing mice. Four test groups were used to mimic clinical protocol: (1) PBS control, (2) DCs + pomalidomide/dexamethasone, (3) Pomalidomide/dexamethasone + PD‐L1 blockade, and (4) DCs + pomalidomide/dexamethasone + PD‐L1 blockade.Results:The combination of DCs + pomalidomide with dexamethasone + PD‐L1 blockade inhibited more strongly tumor growth and prolonged survival of treated mice compared to the other groups. This effect was associated with a significant reduction in immune suppressor cells, such as myeloid‐derived suppressor cells, regulatory T cells, and M2 macrophages, and level of immunosuppressive factors, such as vascular endothelial growth factor, transforming growth factor‐β, and interleukin‐10, and with the significant induction of immune effector cells, such as CD4+ and CD8+ T cells, memory T cells, natural killer (NK) cells, and M1 macrophages, in the spleen and tumor microenvironment. Functional activities of cytotoxic T lymphocytes and NK cells in spleen were also enhanced by the combination of DCs + pomalidomide with dexamethasone + PD‐L1 blockade.Summary/Conclusion:The collective findings in the murine MM model suggest that DC vaccination combined with pomalidomide and PD‐L1 blockade synergistically enhance antitumor immunity through two‐way mechanism, which inhibits immunosuppressive cells while activating effector cells with superior polarization of the Th1/Th2 balance in favor of the tumor immune response.

Ikaros mutations can bypass the requirement for second co-stimulatory signal and lead to break of B-cell tolerance

Abstract Autoimmunity is thought to arise due to a combination of genetic and environmental factors. IKZF1, encoding the Zinc finger (ZnF) transcription factor Ikaros, is implicated in human autoimmunity through GWAS and recent discovery of germline IKZF1 mutations in patients with autoimmune diseases. We have previously created mouse models with targeted deletions of Ikaros DNA-binding ZnF1 or ZnF4, and recently found that mice lacking Ikaros ZnF4 (Ik-dF4) displayed very high levels of autoreactive antibodies and elevated levels of the autoimmunity-associated cytokine IL-6 in serum at young age, while mice lacking ZnF1 did not. Furthermore, ex vivo stimulation of peripheral B cells with anti-IgM (aIgM) +/− CD40L revealed that Ik-dF4 B cells responded to BCR-stimulation alone (aIgM), while wt B cells required a second co-stimulatory signal (CD40L). To understand the underlying mechanism, we performed transcriptome profiling of these stimulated wt and Ik-dF4 B cells. Overall, the Ik-dF4 B cells displayed the same pattern of gene-expression changes as wt B cell upon activation by both signals (aIgM + CD40L). Further analysis revealed a set of genes selectively altered upon aIgM alone in Ik-dF4 B cells, and another set of genes that were aberrantly expressed in Ik-dF4 B cells at baseline. Pathway analysis suggest candidate mediators, and we have initiated functional studies. Preliminary results indicate that a small molecule inhibitor can block the aberrant activation with aIgM alone in Ik-dF4 B cells, while displaying only partial inhibition of the normal two-signal activation. Studies are actively ongoing to confirm these preliminary findings, and we will present our results to date at the meeting for how Ikaros regulates B cell tolerance.

Germline deletion of ETV6 in familial acute lymphoblastic leukemia

AbstractRecent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.

Transcriptome Profiling Analyses Comprehensively Identified Molecular Subgroups of B-Precursor Acute Lymphoblastic Leukemia

▪BackgroundB-precursor acute lymphoblastic leukemia (B-ALL) represents a heterogeneous group of hematological malignancies. Previous studies have identified distinctive gene expression profiles for several molecular subtypes of B-ALL with both biological and clinical importance. However, a proportion of B-ALL have remained poorly characterized with unclear underlying genomic abnormalities. We therefore initated a large-scale international collaborative study to comprehensively reanalyze and delineate the transcriptome landscape of 1,223 B-ALL cases using RNA-seq-driven genomic analyses.MethodsRNA-seq data of 1,223 patients with B-ALL were collected from from Lund University Hospital, the Singapore and Malaysia MaSpore cohort, the Japan Adult Leukemia Study Group (JALSG), Therapeutically Applicable Research to Generate Effective Treatments (TARGET)/Children's Oncology Group(COG) cohort and Multicenter Hematology-Oncology Protocols Evaluation System (M-HOPES) by the Shanghai Institute of Hematology (SIH). We performed gene expression based analyses to identify molecular subtypes and their defining mRNA features. In parallel, we also systematically identified all gene fusions and potential driver mutations in each case. We subsequently defined B-ALL subtype by integrative genomic analysis and comprehensively evaluated their effects on outcomes across B-ALL treatment regimens.ResultsIn this comprehensive analysis of the transcriptomic landscape of 1,223 B-ALL cases, several novel molecular subtypes of B-ALL were identified by strict statistical test capable of scrutinizing subtle genetic features. Totally, fourteen gene expression subgroups (G1-G14) were identified in the integrated B-ALL datasets. Apart from extending eight previously described subgroups (G1-G8 respectively associated to MEF2D fusions, TCF3-PBX1, ETV6-RUNX1/-like, DUX4 fusions, ZNF384 fusions, BCR-ABL1/Ph-like, high hyperdiploidy and MLL fusions), we additionally defined six transcriptome subgroups: G9 associated to both PAX5 and CRLF2 fusions; G10 and G11 respectively to hotspot mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y); G12 to IGH-CEBPE fusion and hotspot mutations in ZEB2 (p.H1038R), while G13 and G14 respectively to TCF3/4-HLF and NUTM1 fusions. We also analyzed the non-silent sequence variants with available WES and RNA-seq data based on an in-house analysis criteria inspired from several published works. We next analyzed non-silent sequence variants in available WES and RNA-seq data based on an in-house analysis criteria inspired by previously published work. We identified 44 genes that were recurrently mutated in at least 1% of the cases (12/1,223 cases). Non-silent variants in NRAS, KRAS, FLT3, KMT2D, PAX5, PTPN11, CREBBP and TP53 exhibited the highest mutation frequencies (3-14%)Conclusion/SummaryLeukemogenic factors contributing to B-ALL are highly heterogeneous. The large-scale cohort transcriptome sequence analysis in B-ALL revealed distinct molecular subgroups that reflect discrete paths of B-ALL, informing disease classification and prognostic stratification. Our work has thus further revealed the complexity of leukemogenesis in B-ALL and suggested the necessity of multiple therapeutic approaches. DisclosuresNo relevant conflicts of interest to declare.

Clinical, Cytogenetic and Immunophenotype Distribution of Adult Acute Lymphoblastic Leukemia Among Latinos — a Report from a Large Single Institution Cohort in Southern California

Introduction:Acute Lymphoblastic Leukemia (ALL) is divided into Pre-B and T phenotypes depending on the progenitor cell giving rise to the malignant clone. This disease is the most common cancer in children while rare in adults. Interestingly, higher incidence rates and poorer outcomes have been reported in patients of Latin American ethnicity. This has been attributed to higher rates of Philadelphia-Like (Ph-like) ALL and increased incidence of high risk features such as absence of hyperdiploidy and absence of t(12;21). The current data among Latinos either exclusively looks at B cell precursor (BCP) ALL or does not distinguish between subtypes. Our study sets out to better define the phenotype of this subset of patients with traditionally poor outcomes.Methods:Our database consisted of 825 adult patients (ages 18-85 years) diagnosed with ALL from the City of Hope Comprehensive Cancer Center in Los Angeles, California. The population in Southern California has a particularly high number of Latinos due to its proximity to Mexico and Central America. According to the 2017 United States Census, 48.6% of the Los Angeles population is of Hispanic or Latino origin. Age at diagnosis, initial WBC count, CNS involvement, imumnophenotype, cytogenetics and rate of hematopoietic cell transplantation during therapy were assessed between ethnic groups. Multivariable Logistic Regression was done to determine the features that distinguish Latino patients for Non-Hispanic Whites.Results:57.4% of the patients were Latinos and 42.6% were Non-Hispanic Whites (NHWs), a distribution unique to the Los Angeles area and consistent with US census population data. BCP ALL was more common in Latinos when compared to NHWs with a Hazard Ratio of 5.13 (95% CI between 2.97-8.87). The presence of the t(9;22) or Philadelphia chromosome (Ph+) was more common in NHW patients. The Hazard Ratio for Ph+ ALL in NHWs was 0.53 (95% CI between 0.37-0.77) CNS involvement was more common in Latino patients and MLL was the only cytogenetic abnormality less common in NHWs. Normal Karyotype (NK), Complex and Others were all more common in Latino patients. No difference was found between WBC counts of Latinos and NHW. (Fig. 1).Discussion:ALL has been shown to have higher rates in the Latino population. Our results among adults ages 18-85 years old, demonstrated that this difference can be attributed to higher incidence of BCP ALL and not T-cell among Latinos. This is consistent with the previously reported higher rate of Ph-Like ALL and distinct molecular aberrations among Latinos. Our data shows that the rate of Ph+ ALL is higher in NHWs when compared to Latinos. This is a very interesting finding as the presence of Ph+ ALL and Ph-like ALL are mutually exclusive. Associations have been previously reported between Ph-like ALL and Latin American ethnicity. Although there are types of ALL that are neither Ph+ nor Ph-like this finding suggests a higher incidence of Ph-like ALL in Latinos. At the present time, standard cytogenetics do not include CRLF2, IKZF1 and other mutations found in Ph-like ALL. Complex cytogenetics and CNS involvement, both factors associated with poor prognosis, are most likely to present in a Latino patient. While the precise mechanism is unknown, our findings further support the hypothesis that a unique genetic fingerprint has led to development and outcomes of Ph-like ALL in Latinos. It is evident that this subset of unfavorable BCP ALL population needs special attention. More studies are imperative to further elucidate the mechanism behind Ph-like ALL and pinpoint the risk factors in the Latino population. [Display omitted] DisclosuresDouer:Spectrum: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Jazz Pharmaceuticals: Consultancy; Gilled Sciences, Inc: Consultancy; Shire: Membership on an entity's Board of Directors or advisory committees.

Germline GAB2 Mutations in Childhood Acute Lymphoblastic Leukemia

Recent studies using next-generation sequencing of selected individuals, such as those with familial leukemia or congenital syndromes, have identified rare and highly penetrant germline mutations that predispose to childhood acute lymphoblastic leukemia (ALL). High hyperdiploidy (HD), the most common cytogenetic subtype of childhood ALL, is enriched in children with RASopathies who develop ALL and, similarly, a high proportion of ALL patients with germline ETV6 or IKZF1 mutations presented with the HD subtype. Here, we aimed to identify novel predisposition genes in a selected group of HD-ALL patients.Targeted sequencing of 538 cancer-relevant genes was carried out using the UCSF500 Cancer Gene Panel in diagnostic bone marrow (i.e. tumor) DNA from 57 HD-ALL patients from the California Childhood Leukemia Study (CCLS). HD-ALL patients were selected based on absence of somatic KRAS or NRAS hotspot mutations detectable by Sanger sequencing, and absence of somatic copy number deletions from multiplex ligation-dependent probe amplification (MLPA) assays. After filtering out likely somatic mutations (mutant allele fraction <0.44), and restricting to variants with low frequency in unselected individuals (allele frequency <0.01% in the Exome Aggregation Consortium, ExAC) and with predicted functional effects (Combined Annotation Dependent Depletion, CADD score ≥20), we identified 151 putative predisposing mutations. Of 41 mutations of interest selected for validation, 37 (90.2%) were confirmed as germline in origin via Sanger sequencing of remission or newborn bloodspot DNA.Rare and predicted functional germline mutations in known (NBN, SH2B3, ETV6, CREBBP, MSH6) or suspected (MLL, ABL1, FLT3, MYH9) ALL predisposition genes were identified in nine out of 57 patients (15.8%). Three additional patients harbored germline mutations in the GRB2-associated binding protein 2 (GAB2), a known binding partner of PTPN11-encoded SHP2 and activator of the ERK/MAPK and PI3K/AKT pathways. Two GAB2 mutations, a missense mutation S592F and frameshift mutation P621fs, were predicted to be highly functional (CADD scores = 34 and 36 respectively) and absent in ExAC. Frequency of rare and damaging GAB2 mutations was significantly higher in our patient set (2.6%) than in ExAC (0.28%, P = 2.70 x 10-6). We replicated this finding in sequencing data from 309 ALL patients in the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project (0.81% vs. 0.28%, P = 0.015).Patient GAB2 mutations were cloned into HEK293 cells and, following EGF stimulation, we found that the P621fs mutation reduced SHP2 binding and ERK1/2 phosphorylation but increased AKT phosphorylation. This suggested possible Ras-independent leukemogenic effects, supported by a lack of somatic Ras pathway mutations in the three GAB2 mutant patients. Additional functional analyses and sequencing of larger patient cohorts will be required to elucidate the role of germline GAB2 mutations in childhood ALL. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional landscape of B cell precursor acute lymphoblastic leukemia based on an international study of 1,223 cases

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3-PBX1 fusions, ETV6-RUNX1-positive/ETV6-RUNX1-like, DUX4 fusions, ZNF384 fusions, BCR-ABL1/Ph-like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH-CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4-HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.