LMO2 Expression Research Articles

High-grade B-cell lymphoma with 11q aberration in the HIV setting: a clinicopathological study of 10 cases and literature review

High-grade B-cell lymphoma with 11q aberration (HGBL-11q) is a distinct lymphoma entity according to the 5th edition of the WHO classification of hematolymphoid tumors. It lacks MYC translocation but carries proximal gains and/or telomeric losses of chromosome 11q. This rare type of B-cell lymphoma is less frequently reported in people living with HIV (PLWH), and its exact frequency remains unclear. Our goal was to retrospectively analyze its frequency in a cohort of aggressive B-cell lymphomas in PLWH, including Burkitt lymphoma (BL, n = 35), diffuse large B-cell lymphoma (DLBCL, n = 48), high-grade B-cell lymphoma, not otherwise specified (HGBL-NOS, n = 13), which was diagnosed as AIDS-related lymphoma (ARL) at our institution. In total, 10/96 (10.4%) cases harbored the typical 11q aberration pattern, predominantly those that had been classified as BL (6/35, 17.1%), DLBCL (2/48, 4.2%), and HGBL, NOS (2/13, 15.4%). We also evaluated 7 cases of AIDS-related HGBL-11q (AR-HGBL-11q) reported in the literature. The median age of our cohort was 35 years, and all the patients were male. Most cases (70%) had a history of HIV infection for over 1 year, and all were involved in lymph nodes (100%), frequently involved extranodal sites (60%), and Ann Arbor stage III/IV. In histomorphology, the cases exhibited diverse cytological features, reminiscent of BL (6 cases), DLBCL (2 cases), and HGBL (2 cases). A comparison of the combined cohort of 17 AR-HGBL-11q cases with 11 ARL cases that lacked both MYC rearrangement and 11q aberration at our institution showed that HGBL-11q cases were characterized by strikingly coarse apoptotic debris (P < 0.001), background rich in eosinophils (P = 0.002), higher expression of the germinal centre marker LMO2 (P = 0.080), lower expression of MUM1 (P = 0.004), BCL2 (P = 0.007), and LEF1 (P = 0.080), and lower positivity for EBER in situ hybridisation (P = 0.027). Notably, one case in our series was EBV-positive, a finding not previously reported in the literature. Furthermore, comparing the prognosis between these two groups, AR-HGBL-11q showed a relatively favorable prognosis (P = 0.15), although the difference was not statistically significant. We analyzed this rare lymphoma entity in the HIV setting and highlighted the importance of integrating histomorphological and immunophenotypic features in its diagnosis and classification.

Chromogenic LMO2 mRNA ISH Expression Correlates with LMO2 Protein and Gene Expression and Captures Their Survival Impact in Diffuse Large B-Cell Lymphoma, NOS.

LMO2 is a relevant gene involved in B-cell ontogeny and a survival predictor of aggressive large B-cell lymphomas (aLBCL). Most studies assessing LMO2 mRNA expression have relied on microarray platforms or qRT-PCR methods, overlooking tissue morphology. In this study, we evaluate LMO2 RNA expression by chromogenic in situ hybridization (CISH) in normal tissue and in a series of 82 aLBCL. LMO2 CISH was performed in formalin-fixed paraffin-embedded tissues, scored by three different methods, and correlated with a transcriptome panel. We obtained statistically significant results correlating the methods of evaluation with LMO2 protein expression and gene expression results. Normal tonsil tissue showed high levels of LMO2, particularly within the light zone of the germinal center. Conversely, in aLBCL, a notable reduction in LMO2 expression was noted, remarkably in cases carrying MYC rearrangements. Furthermore, significant results were obtained through overall survival and Cox regression survival analysis, incorporating International Prognostic Index data alongside LMO2 expression levels. We show a reliable method to identify LMO2 mRNA expression by CISH, effectively capturing many of the reported biologic features of LMO2.

Abstract B052: The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer

Abstract Introduction: Previously we discovered that a novel metastasis-initiating cell (MIC) population is marked by LMO2, a transcriptional adaptor protein in hematopoietic stem cells, and a T-cell oncogene. Knocking down LMO2 did not affect the growth of primary tumors, but did significantly reduce the number of circulating tumor cells and lung metastases. Transcriptional analysis of LMO2 knockdown shows the HALLMARK_DNA_REPAIR pathway is upregulated, suggesting that LMO2 is involved in regulating DNA repair in breast cancer cells. Interestingly, a previous study reported that in diffuse large B-cell lymphoma high LMO2 expression results in defective repair of double-stranded DNA breaks (Parvin et al., Cancer Cell, 36, 3 [2019]). We hypothesize that in breast cancer LMO2 expression is regulating the choice between homology-directed repair (HDR), an error-free method of DNA repair, and non-homologous end-joining (NHEJ), a more error-prone method. Cells that use NHEJ to repair DNA are dependent on poly (ADP-ribose) polymerase (PARP) and can be targeted with PARP inhibitors. LMO2 could thus be used as a new biomarker for benefit from PARP inhibition. Methods: LMO2 was identified using single-cell RNAseq data from human breast cancer patients. In vitro and in vivo assays using breast cancer cell lines and patient-derived xenografts were used to characterize the effect of LMO2 on metastasis. To study the role of LMO2 in the DNA damage response, we used MDA-MB-468 and HCC1806 cells with control and 2 independent shRNAs targeting LMO2. Immunofluorescent staining for γH2AX, 53BP1, and RAD51 was performed to measure changes in DNA damage and repair markers. Control and LMO2 knockdown cells were treated with olaparib, with cellular proliferation measured with a WST-1 assay and colony formation measured with crystal violet staining. Results and Conclusion: Knocking down LMO2 expression results in decreased NHEJ and increased HDR in MDA-MB-468 and HCC1806 cells. There is no significant difference in DNA damage overall as measured by γH2AX foci/nucleus. LMO2 knockdown in MDA-MB-468 cells reduces sensitivity to PARP inhibition with olaparib, as measured by an increase in the IC50 values. Our preliminary data suggest that LMO2 expression likely regulates the choice between NHEJ and HDR. LMO2 expression introduces a bias towards NHEJ, which sensitizes cells to PARP inhibition. This suggests that LMO2+ cells could be targeted with PARP inhibition, which could thus be used to target metastasis-initiating cells. Citation Format: Isobel Fetter, Veronica Haro-Acosta, Shaheen Sikandar. The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B052.

Decoding the genetic symphony: Profiling protein-coding and long noncoding RNA expression in T-acute lymphoblastic leukemia for clinical insights.

T-acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy characterized by the abnormal proliferation of immature T-cell precursors. Despite advances in immunophenotypic classification, understanding the molecular landscape and its impact on patient prognosis remains challenging. In this study, we conducted comprehensive RNA sequencing in a cohort of 35 patients with T-ALL to unravel the intricate transcriptomic profile. Subsequently, we validated the prognostic relevance of 23 targets, encompassing (i) protein-coding genes-BAALC, HHEX, MEF2C, FAT1, LYL1, LMO2, LYN, and TAL1; (ii) epigenetic modifiers-DOT1L, EP300, EML4, RAG1, EZH2, and KDM6A; and (iii) long noncoding RNAs (lncRNAs)-XIST, PCAT18, PCAT14, LINC00202, LINC00461, LINC00648, ST20, MEF2C-AS1, and MALAT1 in an independent cohort of 99 patients with T-ALL. Principal component analysis revealed distinct clusters aligning with immunophenotypic subtypes, providing insights into the molecular heterogeneity of T-ALL. The identified signature genes exhibited associations with clinicopathologic features. Survival analysis uncovered several independent predictors of patient outcomes. Higher expression of MEF2C, BAALC, HHEX, and LYL1 genes emerged as robust indicators of poor overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS). Higher LMO2 expression was correlated with adverse EFS and RFS outcomes. Intriguingly, increased expression of lncRNA ST20 coupled with RAG1 demonstrated a favorable prognostic impact on OS, EFS, and RFS. Conclusively, several hitherto unreported associations of gene expression patterns with clinicopathologic features and prognosis were identified, which may help understand T-ALL's molecular pathogenesis and provide prognostic markers.

Jmjd3 Drives Immature T-Cell Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. Despite an improved survival rate due to intensified chemotherapy and bone marrow transplantation, still 20% of T-ALL patients are primary refractory to the standard treatment or relapse, and have a poor clinical outcome. T-ALL patients can be classified into molecular genetic subgroups based on the expression or genomic alterations of specific oncogenic transcription factors. One such subgroup the early T-cell precursor ALL (ETP-ALL) is a heterogeneous subgroup of immature leukemias that often display high expression of LYL1 and LMO2. It's important to improve our knowledge of T-ALL biology and developing novel targeted anti-TALL therapies. We previously showed that the H3K27 demethylase Jmjd3 plays an important role in T-ALL and is a good candidate for the treatment of T-cell leukemia. Aim: Identify the role of Jmjd3 in the initiation of immature T-ALL. Methods: We generated a conditional Rosa26- Jmjd3 overexpression mouse model (Figure 1A), and crossed it with a hematopoietic-specific Vav-icre mice, to obtain Jmjd3 Vav mice. To investigate the role of Jmjd3 in tumor initiation, we monitored survival of a Jmjd3 Vav cohort. Arising tumors were characterized by immunohistochemistry, flow cytometry, and were transplanted into secondary immunodeficient recipients. To characterize the pre-leukemic compartment, we performed single cell feature barcoding and RNA sequencing. Also bulk RNAseq and proteomics were performed on primary and transplanted Jmjd3-driven tumors. To investigate the role of Jmjd3 in immature T-cell biology in human and mice, we analyzed thymi from 8-weeks-old control and Jmjd3 Vavmice, and performed OP9-DL4 stromal co-cultures with transduced CD34+ human cord blood cells that overexpressed JMJD3, respectively. We used ETP-ALL patient-derived xenografts (PDX) models to validate markers identified in single cell analysis. Results: We found that hematopoietic Jmjd3 overexpression resulted in hematological malignancies with 38% penetrance (n= 8/21) and a median survival of 63 weeks (Figure 1B). Immunohistochemistry confirmed overexpression of Jmjd3, decreased levels of H3K27me3, and increased levels of H3K27ac in Jmjd3 Vav tumors. Immunophenotypic analysis revealed immature Thy1.1 +CD4 -CD8 - cells in all obtained Jmjd3 Vav tumors (Figure 1C), indicative of an immature the early T-cell precursor (ETP)-like ALL. These immature T-ALLs gave rise to primary (7 weeks) and secondary (3 weeks) transplants upon their transplantation in immunocompromised mice. Bulk RNAseq confirmed that the transcriptional feature of the primary tumor were maintained upon transplantation. The role of Jmjd3 in stimulating immature thymocyte development was confirmed in thymi from 8-weeks-old Jmjd3 Vavmice and could be validated as well in human T-cell development using stromal OP9-DL4 co-cultures. Finally, we used single cell sequencing technologies to characterize both transcriptional features and cell-surface markers within the tumor fraction (Figure 1D). We confirmed that the tumor cluster was ETP-like with absence of CD1, CD5 and mature T-cell markers, and expression of CD44, Lyl1, and myeloid markers. Furthermore, we could identify cell-surface in immature Jmjd3-driven tumors that were also present in human T-cell stromal culture and ETP-ALL PDXs. Conclusion: We identified Jmjd3 as a driver of immature thymocyte populations that give rise to ETP-ALL.

Simulated microgravity altered the gene expression profiles and inhibited the proliferation of Kupffer cells in the early phase by downregulating LMO2 and EZH2

Microgravity is a primary challenge that need to overcome, when human travel to space. Our study provided evidence that Kupffer cells (KCs) are sensitive to simulated microgravity (SMG), and no similar research report has been found in the literature. Using transcriptome sequencing technology, it was showed that 631 genes were upregulated and 801 genes were downregulated in KCs after treatment under SMG for 3 days. The GO analysis indicated that the proliferation of KCs was affected when exposed to SMG for 3 days. CCK-8 assay confirmed that the proliferation of KCs was inhibited in the third day under the environment of SMG. Furthermore, we identified 8 key genes that affect the proliferation of KCs and predicted 2 transcription factors (TFs) that regulate the 8 key genes. Significantly, we found that microgravity could affect the expression of LMO2 and EZH2 to reduce the transcription of Racgap1, Ccna2, Nek2, Aurka, Plk1, Haus4, Cdc20, Bub1b, which resulting in the reduction in KCs proliferation. These finding suggested that the inhibition of KCs proliferation under microgravity may influence the homeostasis of liver, and LMO2 and EZH2 can be the targets in management of KCs’ disturbance in the future practice of space medicine.

PARP inhibitor exerts an anti-tumor effect via LMO2 and synergizes with cisplatin in natural killer/T cell lymphoma

BackgroundPARP inhibitor (PARPi), as a kind of DNA damage repair inhibitor, has been shown to be effective in various solid tumors and hematologic malignancies. Natural killer/T cell lymphoma (NKTCL) is a highly aggressive malignancy, the treatment of which has long been a major challenge in the clinic. Here, we investigated the efficacy and mechanism of PARPi, and the therapeutic value of PARPi combined with cisplatin in NKTCL.MethodsThe cell proliferation, cell apoptosis, and cell cycle of NKTCL cells were detected respectively by CCK-8 and flow cytometry. The changes of mRNA expression and protein level were measured respectively by mRNA-sequencing, quantitative real-time PCR, western blotting, and immunofluorescence. LMO2 expression was detected by immunohistochemistry and western blotting. Targeted knockdown of LMO2 was conducted by short hairpin RNA. The tumor xenograft models were established to evaluate the efficacy of drugs in vivo.ResultsPARPi inhibited cell proliferation, promoted cell apoptosis, and induced S-phase cell cycle arrest in NKTCL cells. PARPi led to the accumulation of DNA damage by blocking DNA repair and DNA replication. Additionally, LMO2 deficiency reduced the sensitivity of NKTCL cells to PARPi. Finally, the combination of PARPi and cisplatin exhibited significant synergistic effects both in vitro and in vivo.ConclusionsIn summary, we found that PARPi exerted an anti-tumor effect via LMO2 and synergized with cisplatin in NKTCL, which provides the theoretical basis for the clinical application of PARPi.

Establishment and Clinical Significance of Prognostic Nomogram Model for Diffuse Large B-Cell Lymphoma Based on Immunohistochemistry Markers and International Prognostic Index Scores

To retrospectively analyze clinical characteristics and survival time of patients with diffuse large B-cell lymphoma (DLBCL), detect prognosis-related markers, and establish a nomogram prognostic model of clinical factors combined with biomarkers. One hundred and thirty-seven patients with DLBCL were included in this study from January 2014 to March 2019 in the First Affiliated Hospital of Nanchang University. The expression of GCET1, LMO2, BCL-6, BCL-2 and MYC protein were detected by immunohistochemistry (IHC), then the influences of these proteins on the survival and prognosis of the patients were analyzed. Univariate and multivariate Cox regression analysis were used to gradually screen the prognostic factors in nomogram model. Finally, nomogram model was established according to the result of multivariate analysis. The positive expression of GCET1 protein was more common in patients with Ann Arbor staging I/II (P =0.011). Compared with negative patients, patients with positive expression of LMO2 protein did not often show B symptoms (P =0.042), and could achieve better short-term curative effect (P =0.005). The overall survival (OS) time of patients with positive expression of LMO2 protein was significantly longer than those with negative expression of LMO2 protein (P =0.018), though the expression of LMO2 protein did not correlate with progression-free survival (PFS) (P >0.05). However, the expression of GCET1 protein had no significant correlation with OS and PFS. Multivariate Cox regression analysis showed that nomogram model consisted of 5 prognostic factors, including international prognostic index (IPI), LMO2 protein, BCL-2 protein, MYC protein and rituximab. The C-index applied to the nomogram model for predicting 4-year OS rate was 0.847. Moreover, the calibrated curve of 4-year OS showed that nomogram prediction had good agreement with actual prognosis. The nomogram model incorporating clinical characteristics and IHC biomarkers has good discrimination and calibration, which provides a useful tool for the risk stratification of DLBCL.

Zebrafish ELL-associated factors Eaf1/2 modulate erythropoiesis via regulating gata1a expression and WNT signaling to facilitate hypoxia tolerance

EAF1 and EAF2, the eleven-nineteen lysine-rich leukemia (ELL)-associated factors which can assemble to the super elongation complex (AFF1/4, AF9/ENL, ELL, and P-TEFb), are reported to participate in RNA polymerase II to actively regulate a variety of biological processes, including leukemia and embryogenesis, but whether and how EAF1/2 function in hematopoietic system related hypoxia tolerance during embryogenesis remains unclear. Here, we unveiled that deletion of EAF1/2 (eaf1−/− and eaf2−/−) caused reduction in hypoxia tolerance in zebrafish, leading to reduced erythropoiesis during hematopoietic processes. Meanwhile, eaf1−/− and eaf2−/− mutants showed significant reduction in the expression of key transcriptional regulators scl, lmo2, and gata1a in erythropoiesis at both 24 h post fertilization (hpf) and 72 hpf, with gata1a downregulated while scl and lmo2 upregulated at 14 hpf. Mechanistically, eaf1−/− and eaf2−/− mutants exhibited significant changes in the expression of epigenetic modified histones, with a significant increase in the binding enrichment of modified histone H3K27me3 in gata1a promoter rather than scl and lmo2 promoters. Additionally, eaf1−/− and eaf2−/− mutants exhibited a dynamic expression of canonical WNT/β-catenin signaling during erythropoiesis, with significant reduction in p-β-Catenin level and in the binding enrichment of both scl and lmo2 promoters with the WNT transcriptional factor TCF4 at 24 hpf. These findings demonstrate an important role of Eaf1/2 in erythropoiesis in zebrafish and may have shed some light on regeneration medicine for anemia and related diseases and on molecular basis for fish economic or productive traits, such as growth, disease resistance, hypoxia tolerance, and so on.

LMO2 is essential to maintain the ability of progenitors to differentiate into T-cell lineage in mice.

Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two murine Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.

Loss of CDKN2C Accelerates Progression of Notch-1 Induced T Lymphoblastic Leukemia Via Activation of LMO2

The cyclin-dependent kinases inhibitor CDKN2C (p18INK4C, p18) is a member of the INK4 family that specifically blocks the activity of CDK4/6 in the G1 phase of cell cycle. In the hematopoietic system, deletion of p18 was suggested to be associated with T cell malignancies in mice and B cell malignancies in humans. The mice reconstituted with p18 knockout (ko) bone marrow (BM) cells spontaneously developed acute T lymphoblastic leukemia (T-ALL) in serial transplantation (2 years after transplantation) originated from a CD3lowcell population. However, how p18 is involved in the development of T-ALL leukemia is largely unknown. In this study, Intercellular domain of Notch1 (ICN-1) induced T-ALL model was used to explore the role of p18 deficiency in the development of T-ALL leukemia.Enriched hematopoietic stem and progenitor LSK (Lin- c-Kit+ Sca-1+) cells from p18+/+ or p18-/- mice were transduced with ICN1-GFP retrovirus and the same number of GFP+ cells from each group were sorted and transplanted into lethally irradiated recipient mice. About 50 days later, both groups of recipient mice showed symptom of leukemia and more than 90% donor-derived GFP+ cells were detected in peripheral blood and BM. The phenotypic analysis revealed the CD3 expression in GFP+ cells. The majority of CD3+ cells were CD4/8 double positive in p18+/+ group while that of CD3+ cells were CD4/8 double negative in p18-/- group. Analysis of the thymus revealed that DN3 cells accounted for 70.05±3.75% and 12.38±4.6% of thymocytes in p18-/- group and p18+/+ group, respectively, which have been thought as the leukemia-initiating cells according to a previous report. Serial transplantation was performed to measure self-renewal potential of leukemia cells in sub-lethally irradiated mice. Survival analysis showed an accelerated progression of leukemia in p18-/- group compared with the p18+/+ group in first, secondary and tertiary transplantation (p=0.01, p<0.001, p<0.001 respectively). The cell cycle analysis of leukemia cells by Ki67 assay suggested a decreased proportion of G0/G1 phase and an increased proportion of S/G2/M phase in p18-/- group compared with p18+/+ group. The gene expression analysis also showed higher expression of Notch1 signaling and target genes, such as Hes1 and C-myc in p18-/- leukemia cells. Noticeably, a dramatically increase of LMO2 gene expression was detected in the leukemia cells of p18-/- group compared with p18+/+ group. According to previous studies, as an oncogene, LMO2 plays a critical role in the normal hematopoietic differentiation and is aberrantly expressed in approximately 45% of T-ALL patients. Deletion of p18 activates the expression of LMO2 although a mechanism is yet to be further defined. Taken together, our current study provides evidence and mechanism insights for the role of p18 deficiency during progression of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

T-ALL can evolve to oncogene independence.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

Ldb1 is required for Lmo2 oncogene–induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia

AbstractProlonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Clinical Interest of LMO2 Testing in Aggressive Large B-Cell Lymphomas

Introduction: MYC rearrangements (MYCr) occur in 5 to 15% of diffuse large B-cell lymphomas (DLBCL) and 20 to 35% of high-grade B-cell lymphoma, NOS (HGBL-NOS), are a defining criterion of the category HGBL with rearrangements of MYC and/or BCL2/BCL6 (HGBL, with MYCr and BCL2/BCL6), and may be present in 90% of Burkitt lymphoma. The current WHO classification considers cytogenetic techniques as the appropriate tool to detect MYCr but does not define how to approach to the identification of such alteration. As the global incidence of MYCr in large B-cell lymphomas (LBCL) is low, it is necessary to clarity whether FISH or other cytogenetic methods have to be applied to all LBCL or only in selected cases. We previously identified LMO2 as a potential surrogate marker of MYCr in LBCL (Colomo L, Am J Surg Pathol 2017). Our aim with this study is to confirm this observation and evaluate the clinical impact of this marker in the survival of patients with LBCL. Methods: We have prospectively studied between September 2014 and July 2019 a new series of 180 LBCL including patients with DLBCL, HGBL, with HGBL, with MYCr and BCL2/BCL6, HGBL-NOS and transformed low-grade lymphomas into DLBCL (tDLBCL) diagnosed according to WHO criteria. LMO2 (clone 1A9-1), MYC (clone Y69) and a common immunohistochemistry (IHC) panel of B and T-cell markers have been used for the histological categorization of the cases, using whole tissue sections. The cutoff for LMO2 and MYC were 30% and 40%, respectively. MYC and BCL6 genes were studied using break apart probes, and BCL2 gene using dual-color dual-fusion probes (IGH/BCL2), all from Vysis-Abbott. We have statistically correlated the loss of expression of LMO2 and the overexpression of MYC with the presence or absence of MYCr. Moreover, we performed survival analyses assessing the clinical impact of LMO2 in a series of 162 LBCL patients (112 DLBCL, 20 HGBL, with MYCr and BCL2/BCL6, 4 HGBL-NOS and 26 tDLBCL). The survival series included cases diagnosed before 2014 with IHC and FISH data. Results: The prospective series included 132 patients with DLBCL (78M/52F; median age 67 years, range 35-95), 9 HGBL, with MYCr and BCL2/BCL6 (5M/4F; median age 67 years, range 42-85), 4 HGBL-NOS (2M/2F; median age 58 years, range 42-89), and 35 tDLBCL (31 transformed follicular lymphomas, 3 marginal zone lymphoma and 1 lymphoplasmacytic lymphoma; 23M/20F; median age 64 years, range 40-82). LMO2 and MYC were expressed as follows, respectively: 84/130 (65%) and 46/132 (35%) in DLBCL; 1/9 (11%) and 8/9 (89%) in HGBL, with MYCr and BCL2/BCL6; 0/4 and 3/4 (75%) HGBL-NOS; 25/34 (73%) and 7/33 (21%) tDLBCL. MYCr were identified in 9/132 (7%) DLBCL; all HGBL, with MYCr and BCL2/BCL6; 4/4 HGBL-NOS; 7/35 (20%) tDLBCL. The table shows the comparisons between LMO2 and MYC protein expression for the identification of the presence of MYCr in the series of LBCL. Whereas in the whole series LMO2 and MYC had similar results, among CD10-positive cases, LMO2 had better results than MYC and identified better the presence of MYCr than MYC protein expression. The 5-year progression-free survival (PFS) according the diagnostic categories was 59% for DLBCL, 28% for HGBL, with MYCr and BCL2/BCL6, 25% for HGBL-NOS and 22% for tDLBCL (P=0.015). In addition, PFS was significantly lower for the presence of MYCr (26% vs 53%, P=0.02) and MYC IHC expression (35% vs 53%, P=0.005), and showed a positive trend for LMO2 loss of expression (39% vs 52%, P=0.1). The 5-year overall survival (OS) according the diagnostic categories was 67% for DLBCL, 23% for HGBL, with MYCr and BCL2/BCL6, 50% for HGBL-NOS and 77% for tDLBCL (P&lt;0.001). In addition, OS was significantly shorter for the presence of MYCr (37% vs 71%, P=0.002), MYC protein expression (46% vs 75%, P=0.001), and for LMO2 loss of expression (46% vs 74%, P=0.005). In a Cox regression survival analysis including IPI and LMO2 for the 68 CD10-positive cases, IPI (HR: 1.61 P=0.03) was the most important variable for predicting OS, and LMO2 showed a significant trend (HR: 0.44 P=0.06). However, the addition of MYC IHC and MYCr did not add predictive accuracy to IPI score (HR: 1.6 P=0.31; HR: 1.8 P=0.19, respectively). Conclusions: LMO2 detection by IHC is a useful tool to detect MYCr in aggressive LBCL, particularly in CD10-positive cases. Moreover, LMO2 protein expression captures the prognostic significance of the different diagnostic histological categories and the presence of MYCr in this group of lymphomas. Disclosures Sanchez-Gonzalez: Alexion: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Salar:Celgene: Consultancy; Gilead: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

High LMO2 Expression Confers Sensitivity to PARP Inhibition in DLBCL.

LMO2 expression correlated with increased response to olaparib and olaparib with R-CHOP.

LMO2 as a Biomarker for Hypersensitivity to Genotoxic Therapy

In this issue of Cancer Cell, Parvin and colleagues identify the expression of LMO2 as a biomarker for DNA repair defects in lymphomas. Using isogenic cell lines and xenografts, the authors show that expression of LMO2 predicts sensitivity to PARP inhibition, especially in combination with genotoxic therapy.

LMO2 Confers Synthetic Lethality to PARP Inhibition in DLBCL

Deficiency in DNA double-strand break (DSB) repair mechanisms has been widely exploited for the treatment of different malignances, including homologous recombination (HR)-deficient breast and ovarian cancers. Here we demonstrate that diffuse large B cell lymphomas (DLBCLs) expressing LMO2 protein are functionally deficient in HR-mediated DSB repair. Mechanistically, LMO2 inhibits BRCA1 recruitment to DSBs by interacting with 53BP1 during repair. Similar to BRCA1-deficient cells, LMO2-positive DLBCLs and Tcell acute lymphoblastic leukemia (T-ALL) cells exhibit a high sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Furthermore, chemotherapy and PARP inhibitors synergize to inhibit the growth of LMO2-positive tumors. Together, our results reveal that LMO2 expression predicts HR deficiency and the potential therapeutic use of PARP inhibitors in DLBCL and T-ALL.

Abstract 3836: Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML

Abstract Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p &amp;lt; 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of &amp;lt; 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate &amp;gt; 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values &amp;lt; 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3836.

PS925 LMO2‐INDUCED MURINE T‐CELL ACUTE LYMPHOBLASTIC LEUKEMIA FREQUENTLY EVOLVES TO ONCOGENE INDEPENDENCE

Background:LMO2 is an oncogenic transcription factor that is frequently overexpressed in T‐cell acute lymphoblastic leukemia (T‐ALL) including poor prognosis Early T‐cell Precursor‐like ALL (ETP‐ALL).In transgenic mouse models, LMO2 overexpression causes thymocyte self‐renewal resulting in T‐cell leukemia with long latency. However, the requirement for LMO2 for leukemia maintenance is poorly understood.Aims:1. To develop a regulatable mouse model of LMO2‐induced T‐ALL.2. To determine the molecular features associated with LMO2‐dependence in T‐ALL.Methods:To develop a regulatable model of LMO2‐induced T‐ALL, LMO2 was cloned downstream of the Tetracycline‐regulated element (TRE) promoter (see Figure). To allow us to track LMO2 expression, a Green Fluorescence Protein (GFP) reporter followed by a self‐cleaving peptide (P2A) was cloned upstream of LMO2. This construct was knocked into the Col1a1 locus to generate knock‐in mice (referred to as TRE‐LMO2 mice).To achieve broad overexpression of LMO2 throughout the hematopoietic system, TRE‐LMO2 mice were bred with Vav‐tTA transgenic mice, which express the tetracycline transactivator (tTA) downstream of the pan‐hematopoietic Vav promoter, to generate Vav‐tTA;TRE‐LMO2 mice (referred to as VTL mice), in which LMO2 is overexpressed throughout the hematopoietic system and can be repressed by administration of Doxycycline (Dox) (see Figure).Results:Vav‐tTA;TRE‐LMO2 mice showed widespread LMO2 overexpression throughout the hematopoietic system. This led to a specific block in T‐cell development and to the development of transplantable, self‐renewing T‐cell progenitors in the thymus from a young age, referred to as preleukemic stem cells (pre‐LSCs). This was followed by development of fully penetrant T‐lymphoblastic leukemia resembling human T‐ALL from 5 months of age.In preleukemic mice, Dox‐induced repression of LMO2 rapidly overcame the T‐cell developmental block and eliminated self‐renewing pre‐LSCs in the thymus. In contrast, LMO2 was dispensable for many LMO2‐induced T‐cell leukemias and leukemia‐derived cell lines, implying an evolution of oncogene addiction in many T‐ALLs. LMO2‐dependent T‐ALL correlated with an immature gene expression profile similar to human ETP‐ALL, but could not be predicted by immunophenotype or examination of Notch pathway activation.Summary/Conclusion:We have successfully created a regulatable model of LMO2‐driven murine T‐ALL.imageWhilst continuous LMO2 expression is essential for its preleukemic effects, including T‐cell developmental blockade and thymocyte self‐renewal, LMO2 is frequently dispensable for maintenance of LMO2‐induced murine T‐ALL.LMO2‐dependent murine T‐ALL has an ETP‐like gene expression profile.The regulatable model presented here will be useful to determine the molecular features associated with LMO2‐dependence and critical components of LMO2‐induced self‐renewal pathways in poor prognosis ETP‐ALL.