Relapsed B-ALL Research Articles

TARGET based m6A methylation-related genes predict prognosis relapsed B-cell acute lymphoblastic leukemia

PurposeThe current study aims to investigate the significance of N6-methyladenosine (m6A) methylationrelated genes in the clinical prognosis of childhood relapsed B-cell acute lymphoblastic leukemia (B-ALLL) patient.MethodsTranscriptome data and corresponding clinical data on m6A methylation-related genes (including 20 genes) were obtained from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) database.ResultsThe bone marrow (BM) samples of 134 newly diagnosed (naive) and 116 relapsed B-ALL from TARGET were enrolled in the current study. Three genes (FTO, HNRNPC, RBM15B) showed significant up-regulation in relapsed B-ALL compared with that in naive B-ALL.The three genes had a significantly worse survival (P < 0.05). The LASSO Cox regression model was used to select the most predictive genes as prognostic indicators, and YTHDC1 and FTO were identified as prognostic factors for relapsed B-ALL. Finally, the results of multivariate regression analysis showed that the risk score of m6A methylation-related genes was an independent prognostic factor in relapsed B-ALL (P < 0.05).ConclusionWe found that the expression levels of m6A methylation-related genes were different in naive and relapsed patients with B-ALL and correlated with survival and prognosis.This implies that m6A methylation-related genes may be promising prognostic indicators or therapeutic targets for relapsed B-ALL.

Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics

BackgroundCellular immunotherapy, represented by the chimeric antigen receptor T cell (CAR-T), has exhibited high response rates, durable remission, and safety in vitro and in clinical trials. Unfortunately, anti-CD19 CAR-T (CART-19) treatment alone is prone to relapse and has a particularly poor prognosis in relapsed/refractory (r/r) B-ALL patients. To date, addressing or reducing relapse remains one of the research priorities to achieve broad clinical application.MethodsWe manufactured second generation CART-19 cells and validated their efficacy and safety in vitro and in vivo. Through co-culture of Nalm-6 cells with short-term cultured CART-19 cells, CD19-negative Nalm-6 cells were detected by flow cytometry, and further investigation of the relapsed cells and their resistance mechanisms was evaluated in vitro.ResultsIn this study, we demonstrated that CART-19 cells had enhanced and specific antileukemic activities, and the survival of B-ALL mouse models after CART-19 treatment was significantly prolonged. We then shortened the culture time and applied the serum-free culture to expand CAR-T cells, followed by co-culturing CART-19 cells with Nalm-6 cells. Surprisingly, we observed the proliferation of CD19-negative Nalm-6 cells around 28 days. Identification of potential resistance mechanisms showed that the relapsed cells express truncated CD19 proteins with decreased levels and, more importantly, CAR expression was detected on the relapsed cell surface, which may ultimately keep them antigen-negative. Furthermore, it was validated that CART-22 and tandem CART-22/19 cells could effectively kill the relapsed cells, but neither could completely eradicate them.ConclusionsWe successfully generated CART-19 cells and obtained a CD19-negative refractory relapsed B-ALL cell line, providing new insights into the underlying mechanisms of resistance and a new in vitro model for the treatment of r/r B-ALL patients with low antigen density.

Venetoclax+Azacytidine Followed By Modified BuCy Conditioning Regimen for High Risk or Refractory/Relapsed Acute Lymphoblastic Leukemia and High Risk Myelodysplastic Syndromes

Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only potential curable treatment for myelodysplastic syndromes (MDS) and acute lymphoblastic leukemia (ALL). However, for patients with high-risk or refractory relapsed (R/R) B-ALL with allo-HSCT, the three-year overall survival (OS) rate is only 5-10%, more worse in T-ALL. It is widely known that MDS patients in the high-risk group do not benefit from induction chemotherapy, the five-year OS rate is 23-39% when they received allo-HSCT. Nevertheless, relapse remains a significant cause of treatment failure after allo-HSCT, highlighting the urgent need for improved conditioning regimens that can improve prognosis and reduce relapse rates. Azacytidine (AZA) combined with Venetoclax (VEN), showed a synergistic anti-tumor activity against several hematological malignancies, Therefore, we explored AZA+VEN (VA) combined with modified BuCy(mBuCy) (semustine, cytarabine, busulfan, and cyclophosphamide) conditioning regimen and assessed the safety and effectiveness. Aims ：The aim of this study is to assess the safety, and effectiveness of the VA treatment regimen followed by mBuCy as conditioning regimen for high-risk or R/R ALL(NCT05809167) and high-risk MDS(NCT03256071) . Methods:Patients diagnosed with high-risk or R/R ALL or high-risk MDS (IPSS-R score), undergoing VA+mBuCy as conditioning regimen, and consolidation treatment with demethylating drugs every three months after transplantation, for a total of 8-12 cycles. Results:Patients diagnosed with high-risk or R/R ALL(n=11) or high-risk MDS (IPSS-R score)(n=8) were enrolled between January 13, 2022, and June 9, 2023. The ALL cohort comprised of 5 Ph-negative B-ALL, 2 Ph-positive B-ALL, 3 mixed phenotype leukemias (MPAL), and one T-lymphoblastic lymphoma/leukemia (T-ALL/LBL). There were 7 males and 4 females, with a median age of 33 years (17-54 years) (Table 1). Out of the eleven patients, four had previously undergone Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) therapy, and one patient had received Blinatumomab. One patient experienced relapse, another patient was refractory, and 3 patients had extramedullary infiltration. All patients achieved morphological complete remission prior to transplantation, and 10 patients (90.9%) achieved a MRD negative remission. Additionally, all the transplantations successfully completed, with a median time to absolute neutrophil counts (ANC) recovery of 12 days (11-15 days) and platelet (PLT) recovery of 16 days (15-21 days). The median follow-up time was 6.4 months (2.4 -13.6 months), no patient experienced relapse or death, the OS and DFS are both 100%. There were no grade 3 or higher adverse events (AEs), hepatic veno-occlusive disease (VOD),and the transplant-related mortality (TRM). Four patients who developed acute graft-versus-host disease (aGVHD) post-transplantation, all cases involved grade I/II skin aGVHD, and one of them involved grade III GI aGVHD, and no chronic graft-versus-host disease (cGVHD) were observed during the follow-up period. By the endpoint of follow-up, Cytomegalovirus (CMV) activation was 45.4% (5/11) and Epstein-Barr virus (EBV) activation was 27.3% (3/11). The MDS cohort consisted of 7 cases of MDS-EB-I, 1 case of MDS-EB-II. There were 5 males and 3 females, with a median age of 42 years (23-57 years)(Table 2). Two patients received chemotherapy before transplantation, and the other six patients directly received allo-HSCT. In MDS cohort, the median recovery time for ANC was 12 days (11-13 days) and PLT was 17.5 days (16-20 days). The median follow-up was 14.3 months (1.2-18.3 months), no patient had relapsed or died, both OS and DFS are 100%. There were no grade 3 or higher AEs, and TRM. One patient developed VOD. Three patients developed aGVHD after transplantation, all involving degree I/II skin aGVHD, and there was one case of cGVHD during the follow-up period, where the patient developed a mild pulmonary cGVHD. By the endpoint of the follow-up, the rate of CMV activation was 37.5% (3/8) and EBV activation was 25% (2/8). Conclusion:The VA combined with the mBuCy proves to be a effective and safe conditioning regimen for high-risk ALL and MDS patients. However, more patients are needed for follow-up and validation.

Targeting Bone Marrow Microenvironment for Treatment of High-Risk B-Cell Acute Lymphoblastic Leukemia

Introduction B-cell acute lymphoblastic leukemia (B-ALL) is the most common form of cancer in children, accounting for approximately 20% of all pediatric cancers. Although treatment of B-ALL represents one of the success stories of modern medicine with 5-year survival rates approaching 90%, some children continue to have a dismal prognosis, particularly those with high-risk genetic alterations in their ALL cells or those who relapse. The leukemia microenvironment is an attractive novel therapeutic target, especially for children with high-risk leukemia as dose-limiting toxicities of conventional chemotherapeutic agents have prevented further improvements in survival. Aims In this study, we investigate whether therapeutically targeting the bone marrow (BM) osteoclasts can improve treatment outcome for B-ALL and the mechanisms of crosstalk between osteoclasts and leukemia cells. Methods We have established syngeneic and patient-derived xenograft (PDX) models for high-risk B- ALL that recapitulate the clinical symptoms of bone loss. For in vitro experiments, leukemia cells were cultured with osteoclast-derived conditioned media (CM) for 3 days. Results To evaluate the therapeutic potential of targeting osteoclasts during leukemia development, we administered zoledronic acid (2ug 5 days/week for 2 weeks), a bisphosphonate inhibitor of osteoclastic bone resorption, alone and in combination with imatinib (100mg/kg daily for 28 days) or dasatinib (10mg/kg daily for 28 days) in our BCR-ABL1 + B-ALL syngeneic mouse model. We showed that zoledronic acid treatment significantly reversed leukemia-induced bone loss by attenuating osteoclast activity. Zoledronic acid alone was found to enhance event-free survival in leukemia-bearing mice and further improvement was shown in combination with imatinib (p&amp;lt;0.05) or dasatinib (p&amp;lt;0.01). In addition, we administered zoledronic acid alone and in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in a relapsed B-ALL PDX model. We further demonstrated that zoledronic acid alone or in combination with VXL improved survival compared to mice treated with vehicle (p&amp;lt;0.001) or VXL alone (p&amp;lt;0.01), respectively. In the clinical setting, we assessed the feasibility and safety of incorporating zoledronic acid early into leukemia therapy in three children with B-ALL. The patients were undergoing induction or consolidation chemotherapy and were administered zoledronic acid at the same time. Zoledronic acid was well tolerated without any significant adverse effects, indicating the feasibility of safely administering zoledronic acid in combination with chemotherapy in children with ALL. To gain mechanistic insight into the effect of osteoclasts on leukemia cells, we compared the molecular profile of the leukemia cells cultured with CM and control media using RNA sequencing. There were 30 differentially expressed genes identified at the 5% false discovery rate with a log fold-change value ≥1 or ≤-1. KEGG pathway analysis revealed a significant difference in cell cycle pathway in leukemia cells cultured with CM compared to control. Biological process analysis showed a positive regulation of B-cell proliferation and negative regulation of lymphocyte apoptotic process in leukemia cells cultured with CM compared to control. To functionally examine the effect of osteoclasts on leukemia cells, we showed that osteoclast-derived CM significantly increased B-ALL cell proliferation, as demonstrated by increased cell number (p&amp;lt;0.001), as well as reduced cell percentages in G0/G1 phases and increased S+G2/M phases (p&amp;lt;0.05) via cell cycle analyses. Using annexin V and DAPI staining, we further demonstrated that there were more live cells and less apoptotic and dead cells when the leukemia cells were cultured with CM compared to control (p&amp;lt;0.001). Together, our findings suggest that soluble factors produced by osteoclasts promote leukemia cell proliferation and survival. Conclusion Our study reveals osteoclasts play a role in leukemogenesis and leukemia-induced bone loss, and that targeting osteoclasts can provide dual clinical benefit in terms of bone health and improved survival for children with high-risk B-ALL.

Abstract LB077: Analysis of indel and structural variant error profiles in deep next generation sequencing data

Abstract Background: Accurate detection of low frequency mutations is of critical importance in the study of genetic heterogeneity, such as on the detection of minimal residual diseases for leukemias. Our prior work has resulted in successful error suppression for substitutions (10−4-10−5). However, the error profiles of indels and structural variants (SVs) remain elusive. Results: In this work, we generated ultra-deep sequencing data using our previously established dilution models (COLO829) on known somatic indels (n=23) and SVs (n=17). We discovered that the error rate of indels (10−6) and SVs (&amp;lt;2 × 10−7) are 100- to &amp;gt;1000-fold lower than that of SNVs. This finding was fully recapitulated in our analysis of 347 indels and 1248 SVs discovered from a relapsed B-ALL cohort of 103 patients, although homopolymer indels can have high error rates (&amp;gt;1%). We then performed a comprehensive study of homopolymer indels in 361 cancer driver genes by using whole genome data from 1662 healthy donors from the SJLIFE cohort. Our data indicated that the number of repeating units are highly predictive relative to the error rate of homopolymer indels (R2=0.988, p=4.89 × 10−8). Utilizing these insights, we assayed end-of-induction remission samples from 72 B-cell lymphoblastic leukemia patients that relapsed by selecting ~5 somatic clonal SNV/Indel/SV markers, which confirmed that SVs and indels have &amp;gt;10-fold lower error rates than SNVs. Our next generation sequencing (NGS) approach had 44 positive detections (61%) and outperformed the current standard method of clinical flow cytometry (n=37; 51%) for detecting minimal residual disease. The NGS-based method detected 92% of designed markers for samples with MRD&amp;gt;0.3%, and this detection rate dropped to 27% for MRD between 0.1% and 0.01%, indicating the difficulty in recovering mutant molecules when their frequencies are very low. Conclusions: Overall, we established indel and SV error profiles in deep next generation sequencing data enabling superior tumor detection performance at very low burdens, with lower error rates than what is observed for SNVs. Our work will have a significant impact on the clinical diagnosis and monitoring of human cancers and beyond. Citation Format: Ying Shao, Quang Tran, Pandurang Kolekar, Yanling Liu, Andrea McBride, Tyler Jones, Heather Mulder, Lingyun Ji, Benjamin Huang, Soheil Meshinchi, Jeffery Klco, Jinghui Zhang, William Carroll, Mignon Loh, Patrick Brown, John Easton, Xiaotu Ma. Analysis of indel and structural variant error profiles in deep next generation sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB077.

The Proliferating Cell Nuclear Antigen (PCNA) Transcript Variants as Potential Relapse Markers in B-Cell Acute Lymphoblastic Leukemia.

Leukemia is the most common childhood malignancy in Mexico, representing more than 50% of all childhood cancers. Although treatment leads to a survival of up to 90% in developing countries, in our country, it is less than 65%. Additionally, ~30% of patients relapse with poor prognosis. Alternative splicing plays an important role in transcriptome diversity and cellular biology. This mechanism promotes an increase in the assortment of proteins with potentially distinct functions from a single gene. The proliferating cell nuclear antigen (PCNA) gene encodes two transcripts for the same protein of 261 amino acids, which is associated with several important cellular processes and with several types of cancer. However, the diversity of the transcript variants expressed in this condition is not clear. Then, we used microarray gene expression to identify changes in the exon expression level of PCNA. The data were validated using RT-PCR and Sanger sequencing, and three additional transcripts (PCNA_V3, PCNA_V4, and PCNA_V5) were identified. Computational analyses were used to determine the potential proteins resulting, their structure, and interactions with PCNA native protein and themselves. Additionally, the PCNA transcript variants were inhibited using specific siRNA, determining that their inhibition contributes to the malignant characteristics in vitro. Finally, we quantified the PCNA transcript variants in acute lymphoblastic leukemia samples and identified their expression in this disease. Based on the clinical characteristics, we determined that PCNA_V2 and PCNA_V4 are expressed at significantly low levels in relapsed B-ALL patients. We conclude that the low expression of PCNA_V2 and PCNA_V4 could be a potential molecular marker of relapse in acute lymphoblastic leukemia patients.

S100A8 inhibition in leukemic lymphoblasts induces sensitivity to chemotherapy and inhibition of disease relapse.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and relapsed B-ALL is the leading cause of mortality in children with leukemia due to a lack of response to treatment. S100A8 is a low molecular weight calcium-binding intracellular protein that is expressed in certain cells, and its increased expression is seen in most tumors as well as in relapsed childhood B-ALL cases. The present study indicates the important role of S100A8 in improving viability and resistance to chemotherapy in relapsed B-ALL lymphoblasts. S100A8 levels were compared in B-ALL and relapsed B-ALL lymphoblasts that were sensitive and resistant to Vincristine, respectively. S100A8 was inhibited in the lymphoblasts of two patients by antisense locked nucleic acid (LNA) GapmeRs and the decreased expression of S100A8 was evaluated using quantitative real-time PCR and ELISA. Then, the S100A8 antisense LNA GapmeRs-transfected cells were treated with Vincristine and the expression levels of S100A8 mRNA and S100A8 protein were re-determined. At all of these stages, cell viability and LC50 were assessed by MTT assay. The results showed that S100A8 levels in relapsed B-ALL lymphoblasts were significantly higher than B-ALL lymphoblasts. Moreover, the increase in S100A8 expression was proportionate to the increase in Vincristine resistance in these cells. The S100A8 knockdown procedure using antisense LNA GapmeRs decreased the cell viability and increased vincristine sensitivity in lymphoblasts of two patients, and it also increased the sensitivity to chemotherapy in relapsed B-ALL lymphoblasts. According to the findings of the present study, S100A8 is effective in developing lymphoblast resistance to chemotherapy, and its enhanced expression may contribute to shifting B-ALL into the relapse phase of the illness. As a result, S100A8 may be a valuable target for managing and improving relapses B-ALL.

Dual CD19/CD22 CAR T Cells Show Feasibility in Pediatric/Young Adult B-ALL.

Dual-targeting CAR T cells show safety and efficacy in pediatric/young adult relapsed or refractory B-ALL.

Treating relapsed B-all in in children with mitoxantrone based intensive chemotherapy protocol (TMH rall protocol)-experience from Tata Memorial Hospital

Treating relapsed B-all in in children with mitoxantrone based intensive chemotherapy protocol (TMH rall protocol)-experience from Tata Memorial Hospital

Peripheral leukemia burden at time of apheresis negatively affects the clinical efficacy of CART19 in refractory or relapsed B-ALL

Our previous clinical study achieved complete remission (CR) rates of >90% following chimeric antigen receptor T cells targeting CD19 (CART19) treatment of refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL); however, the influence of the leukemia burden in peripheral blood (PB) blasts remains unclear. Here, we retrospectively analyzed 143 patients treated with CART19 (including 36 patients with PB blasts) to evaluate the effect of peripheral leukemia burden at the time of apheresis. One hundred seventeen patients with high disease burdens achieved 91.5% CR or incomplete count recovery CR and 86.3% minimal residual disease-negative CR, and 26 patients with low disease burdens obtained 96.2% MRD− CR. Collectively, 9 of 36 (25%) patients with PB blasts and 2 of 107 (1.87%) patients without PB blasts did not respond to CART19 therapy. The leukemia burden in PB negatively influenced ex vivo cell characteristics, including the transduction efficiency of CD3+ T cells and their fold expansion, and in vivo cell dynamics, including peak CART19 proportion and absolute count, fold expansion, and persistence duration. Further studies showed that these patients had higher programmed death-1 expression in CART19 products. Our data imply that PB blasts negatively affected CART19 production and the clinical efficacy of CART19 therapy in patients with r/r B-ALL.

Orbital Swelling: An Unusual Case of Relapsed Acute Lymphoblastic Leukaemia in A Preschool Child

Acute lymphoblastic leukaemia (ALL) is the most frequent childhood cancer. Children usually present with signs of bone marrow failure like recurrent or prolonged fever, pallor, lethargy, bleeding tendencies, bone pain and others. Occasionally they may present with sign of infiltration of leukaemic cells into other organs such as testicular and central nervous system, rarely to the periorbital or orbital region. Similarly in relapse cases, they typically presented either in bone marrow, central nervous system relapse or testicular but rarely orbital involvement. Here we report the clinical case of a five-year-old boy who developed relapsed B-ALL, presented to us with unilateral right eye swelling without other clinical findings and absence of blast cells in the peripheral blood film as well as bone marrow aspirate specimen.

Constitutive Activation of Ras and Inactivation of Ink4a/Arf Allele Induce a Fully Penetrant B-Cell Acute Lymphoblastic Leukemia Phenotype in Mice

Despite recent advances in treatment, precursor-B-cell acute lymphoblastic leukemia (B-ALL) remains a challenging clinical entity. Recent genome-wide studies have uncovered frequent genetic alterations involving activating RAS pathway mutations and loss of the INK4A/ARF locus suggesting their important role in the pathogenesis, relapse, and chemoresistance of B-ALL. Therefore, to better understand the oncogenic mechanisms by which these alterations might promote B-ALL and to develop an in vivo preclinical model of relapsed B-ALL, we engineered mouse strains with induced somatic KrasG12D pathway activation and/or loss of Ink4a/Arf during early stages of B-cell development.Pathologic examination revealed significantly enlarged spleens and increased numbers of mononuclear cells in the spleens of 8-week-old KrasG12D mutated animals with or without Ink4a/Arf inactivation, but obvious histologic and IHC differences in spleens and lymph nodes between the mutant genotypes and CD19Cre/+ controls were not found. However, in agreement with the gross pathologic examination, an increase was noted by flow cytometry in splenic B220+ B-cells of mice with the KrasG12D mutation as compared with CD19Cre/+ and CD19Cre/+;Ink4a/ArfL/+ mice. In keeping with this, a significant increase in number of proliferating (Ki-67+) cells within spleen B-cell areas of CD19Cre/+;KrasG12D/+, and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice were observed in comparison with CD19Cre/+ controls and CD19Cre/+;Ink4a/ArfL/+ mice. TUNEL staining revealed fewer apoptotic cells within germinal center B-cell areas in CD19Cre/+;KrasG12D/+, CD19Cre/+;Ink4a/ArfL/+, and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice as compared with CD19Cre/+ controls. Next, to identify the biological processes underlying the observed phenotypical changes, we performed gene-set enrichment analysis of microarray expression data from mutant compared to control mice. In CD19+ B-cells from both CD19Cre/+;KrasG12D/+ and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice, marked upregulation of gene signatures related to proliferation, Ras-related signaling pathways, apoptosis, rewired cellular metabolism, DNA repair, and pre-B stage of B-cell development were observed in both CD19Cre/+;KrasG12D/+ and CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice. Consistent with our previous observations, the proliferative signature in CD19+ B-cells from CD19Cre/+;Ink4a/ArfL/+ mice was less pronounced; instead, upregulation of several signaling pathways related to independence of external growth signals and retention of “stemness” was noted. Although constitutive activation of KrasG12D in B-cells induced prominent transcriptional changes that resulted in enhanced proliferation, it was insufficient by itself to induce development of a high-grade leukemia/lymphoma. Instead, in 40% of mice these engineered mutations promoted development of a clonal low-grade lymphoproliferative disorder resembling human extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue or lymphoplasmacytic lymphoma. Interestingly, loss of the Ink4a /Arf locus, apart from reducing the number of apoptotic B-cells broadly attenuated KrasG12D-induced transcriptional signatures. However, combinedKras activation and Ink4a/Arf inactivation cooperated functionally to induce a fully penetrant, highly aggressive B-ALL phenotype. Cross-species bioinformatic analyses consistently demonstrated that B-ALL tumors in the CD19Cre/+;KrasG12D/+;Ink4a/ArfL/+ mice recapitulated gene expression programs found in a poor-prognosis subgroups of human B-ALL that harbor BCR-ABL and CRLF2 rearrangements. Moreover, 90% of examined murine B-ALL tumors showed loss of the wild-type Ink4a/Arf locus without acquisition of common cooperating events, underscoring the role of Ink4a/Arf in restraining Kras-driven oncogenesis in the lymphoid compartment in our model.Taken together, the functional cooperation and the negative impact on survival of concurrent Ras mutation and Ink/Arf deletion in mice suggest that these changes may also have a functional impact in human B-ALL and should be considered in the course of disease classification, as well as for prognosis and choice of therapeutic strategy. DisclosuresSharpless:G1 Therapeutics: Equity Ownership; Pfizer: Other: NA; HealthSpan Diagnostics: Other: NA; Unity Biotechnology: Other: NA.

Epigenetic analysis reveals significant differential expression of miR-378C and miR-128-2-5p in a cohort of relapsed pediatric B-acute lymphoblastic leukemia cases.

Epigenetic changes play a major role in mediating chemoresistance and relapse in pediatric ALL, and hence in current pilot study, we tried to identify DNA methylation, miRNA expression, and copy number variations (CNVs) in a cohort of relapse pediatric B-ALL cases. DNA methylation, miRNA expression, and CNV analysis were performed in a total of 14, 16, and 18 cases as diagnosis-relapse samples. Briefly, DNA methylation was performed using Infinium HumanMethylation850 chip and data analyzed using RnBeads. miRNA was sequenced on illumina NextSeq500 platform for 20M 75bp SE reads and analyzed by DESeq2. CNVs were assessed by MLPA assay using the ALL P-335 probemix kit and analyzed by coffalyzer.net. On methylation analysis, oncogenes MYCN, MYB, and EGFR and tumor suppressor genes MDM4 & BCL11B were found differentially expressed as compared to controls (p-0.03). In addition, protooncogenes-AXL, HCK, MED12, and ETS2-were hypomethylated/overexpressed in 4 or more cases (P<.05). miRNA analysis revealed significant differential expression of miR-128-2-5p and miR-378C (p-4.4e-15 and p-6.4E-12) in relapse samples. CNV analysis revealed that frequency of good and intermediate/poor risk CNV profile at diagnosis was nearly equal (40% vs 60%). However, CDKN2A/2B and IKZF1 gene CNVs if present in initial diagnostic clone usually persisted in relapse clone. Our pilot study highlights two miRNAs (miR-128-2-5p and miR-378C) as possible candidate biomarkers of relapsed B-ALL. However, these miRNAs and hypomethylated protooncogene signature noted in our data needs validation in a larger series of B-ALL.

Cytogenetic Subgroups Drive Risk Stratification and Response to Chemotherapy and Blinatumomab in Children and Young Adults with Relapsed B-ALL: A Children's Oncology Group Study

Background At initial diagnosis, sentinel cytogenetic (CG) abnormalities differ based on demographic/clinical features and are predictive of relapse risk in B-lymphoblastic leukemia (B-ALL). The relevance of CG at 1st relapse is not well defined. Children's Oncology Group (COG) AALL1331 enrolled non-BCR-ABL1+, non-Down syndrome 1st relapse B-ALL patients (pts) 1-30 years (yr) old. After a common chemotherapy induction (Block 1), pts were risk assigned as: High risk (HR): Early bone marrow (BM) relapse &amp;lt;36 months from diagnosis or isolated extramedullary (IEM) relapse &amp;lt;18 months; Intermediate risk (IR): Late BM relapse (&amp;gt;36 months) with minimal residual disease (MRD) ≥0.1% at end Block-1 (EB-1); Low risk (LR): Late BM relapse with MRD &amp;lt;0.1% at EB-1; Treatment failure (TF): M3 BM or failure to clear central nervous system at EB-1. The primary objective of the trial for HR/IR pts was to randomly compare post-reinduction therapy with 2 blocks of chemotherapy vs. blinatumomab, followed by allogeneic transplant. We previously reported the superiority of blinatumomab for HR/IR pts (Brown, ASH 2019). We hypothesized that CG would correlate with clinical features at relapse and response to therapy, enhancing relapse risk stratification. Methods CG data (karyotype and FISH) at relapse were centrally reviewed. CG groups were defined as: favorable (fav) (double trisomies (DT) or ETV6-RUNX1), unfavorable (unfav) (hypodiploid, KMT2A-rearranged (KMT2A-R) or iAMP21), and neutral (all others). Of 662 eligible pts, 130 IEM relapses were excluded, since CG data were not available due to lack of BM involvement. Of 532 BM relapses, CG data were available for 394 (74%); this group was used for associations of CG with clinical features and response to Block 1. Of 188 BM relapses risk assigned HR/IR and randomized, CG data were available for 147 (78%; 72 Arm A chemotherapy, 75 Arm B blinatumomab); this group was used for associations of CG with response to randomized post-reinduction therapy. CG distributions at initial diagnosis were based on data from over 16,000 pts enrolled on COG upfront trials from 1996-2014. Statistical comparisons used Pearson's chi-squared, Fisher's exact, Wilcoxon rank sum, or logrank test as appropriate. All p values are two-sided. Results As expected, unfav CG were more common at relapse compared to diagnosis (17% vs 7% p&amp;lt;0.001), and the opposite for fav CG (22% vs 42% p&amp;lt;0.001). Also expected was the younger age at relapse for the fav (median 8 yr) and KMT2A-R (median 2 yr) CG vs. others (median 12 yr, p=0.002). WBC at relapse was higher in the KMT2A-R (median 11k/uL), but similar in all others (median 5k/uL, p=0.041). Time from diagnosis to relapse differed by CG group, with late BM relapses comprising 79% of fav vs. 61% of neutral vs. 48% of unfav (p&amp;lt;0.001). EB-1 MRD-negative rates were highest for fav (53%), intermediate for neutral (38%) and lowest for unfav (25%, p=0.002). Interestingly, in fav CG group, 62% of ETV6-RUNX1 vs. 40% of DT were EB-1 MRD-negative (p=0.043), suggesting that as for newly diagnosed pts, MRD clearance during relapse reinduction may be slower for DT than for ETV6-RUNX1. EB-1 risk assignments differed significantly by CG group, with rates for fav/neutral/unfav CG of 57%/35%/27% for LR, 16%/31%/34% for HR, and 3%/7%/7% for TF (p&amp;lt;0.01). After 1st course of randomized therapy for HR/IR pts, the rate of EB-2 MRD negativity was (as previously reported) much higher for arm B (75%) vs arm A (25%, p&amp;lt;0.001). Interestingly, EB-2 MRD clearance rates did not vary significantly by CG group for either randomized arm. There was, however, an influence of CG group on survival for arm B, but not for arm A. The 2 yr DFS/OS for fav CG on arm B was 77%/93%, vs. 49%/65% for neutral and 49%/49% for unfav (p=0.069 for DFS, p=0.035 for OS, univariate analysis). For arm A, DFS/OS was similar and consistently poor in all three groups (44%/52% fav vs. 49%/61% neutral and 39%/61% unfav, p=0.92 for DFS, p=0.62 for OS) (Table 1). Conclusions Pts with fav CG B-ALL relapse later and are more likely to achieve MRD negativity after reinduction chemotherapy, resulting in higher rates of LR risk assignment. Interestingly, while the superior MRD clearance with blinatumomab vs. chemotherapy is seen in all CG groups, this only translated into higher rates of DFS/OS in the fav CG group. Thus, the influence of CG on outcomes after 1st relapse may differ based on whether chemotherapy or immunotherapy is used as post-reinduction consolidation. Disclosures Gore: Amgen, Novartis, Roche: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria. Loh:Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: Institutional Research Funding. Brown:Jazz: Honoraria; Servier: Honoraria; Janssen: Consultancy; Novartis: Consultancy.

Clinical utilization of blinatumomab and inotuzumab immunotherapy in children with relapsed or refractory B-acute lymphoblastic leukemia.

The treatment paradigm for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (rrALL) has been revolutionized given recent clinical trials demonstrating remarkable success of immunotherapies and leading to drug approvals by United States and European agencies. We report experience with commercial blinatumomab and inotuzumab use at two North American pediatric oncology centers in children and adolescents/young adults with B-ALL. Patients 0-25 years old treated with the CD19 × CD3 bispecific T cell-engaging antibody blinatumomab and/or the CD22 antibody-drug conjugate inotuzumab from January 1, 2010, to June 1, 2018, were eligible. Disease status included relapsed B-ALL in second or greater relapse, primary chemotherapy-refractory B-ALL, or B-ALL complicated by severe infection precluding delivery of conventional chemotherapy. We identified 27 patients who received blinatumomab and/or inotuzumab outside of clinical trials during the study period. Four of the 13 patients (31%) with relapsed disease achieved minimal residual disease (MRD)-negative remission, and five patients (39%) underwent hematopoietic stem cell transplant (HSCT). In the 12 patients with primary chemorefractory B-ALL treated with immunotherapy, 11 (92%) achieved MRD-negative remission as assessed by flow cytometry; 10 patients (83%) underwent subsequent HSCT. Two patients with B-ALL in MRD-negative remission received blinatumomab due to severe infection and remained in remission after chemotherapy continuation. Blinatumomab and inotuzumab can induce deep remissions in patients with rrALL and facilitate subsequent HSCT or other cellular therapies. Blinatumomab can also serve as an effective bridging therapy during severe infection. The optimal timing, choice of immunotherapeutic agent(s), and duration of responses require further investigation via larger-scale clinical trials.

Clinical Factors Associated with Improved Survival Following Allogeneic HSCT after CD19 CAR Therapy in Adult Patients with Relapsed B-ALL

Introduction: Autologous chimeric antigen receptor (CAR) T cell therapy has shown to be effective in CD19+ relapsed or refractory (R/R) B-ALL patients (pts). However, relapses are common and the role of post-CAR allogeneic hematopoietic stem cell transplant (alloHSCT) remains unclear, particularly in adults with relapsed B-ALL. We previously reported that there was no survival benefit with post-CAR alloHSCT in overall adult ALL population (Park J et al. NEJM 2018), but it is unclear whether there is a subset of pts who may benefit the most from post-CAR alloHSCT. Therefore, we sought to identify clinical features that are associated with a better long-term survival in adult B-ALL pts treated with CD19 CAR T cells followed by alloHSCT. Methods: We performed a retrospective chart review of adult pts with R/R ALL treated with 19-28z CAR T therapy at MSKCC (NCT01044069), with a particular focus on pts who achieved minimal residual disease (MRD) negative CR to the CAR therapy and subsequently proceeded to alloHSCT. Clinical data was updated with the data cutoff date of July 15, 2019. We examined the association between overall survival and the following clinical factors: impact of age, prior HSCT status, presence of Philadelphia chromosome (Ph), disease status at the time of CAR therapy (MRD vs. morphologic), severity of cytokine release syndrome (CRS) and neurotoxicity (NTX) experienced during CAR therapy. Survival was calculated from the time of CAR T cell infusion. Continuous variables were analyzed univariately using cox PH models. Categorical variables were analyzed using Kaplan Meier methodology and log rank tests. Results: Of the 53 pts who received 1928z CAR T cells, 16 pts proceeded to alloHSCT after achieving MRD negative CR to the CAR therapy. The median age of these pts was 38 (range, 23-66), and baseline disease and pt characteristics as well as response and toxicity to CAR T therapy are listed in Table 1. Of the 16 pts, 6 pts remain alive in remission; 4 pts died of relapse (median time to relapse: 104.5 days, 57-194), and 6 pts died of transplant-related mortality (TRM) in remission (n=4, infection; n=1, pulmonary VOD; n=1, respiratory failure of unclear etiology). Detailed disease and treatment characteristics by the outcome categories are listed in Table 2. For those pts who are alive in remission, the median survival is 47.5 months (range, 39.7-92.4). Among the examined clinical variables, we found younger age and no severe NTX (i.e. grade 0-2) after CAR therapy to be significantly associated with improved overall survival (p=0.014 and 0.05, respectively) but prior lines of therapy, time to HSCT, prior HSCT, Ph chromosome, disease status at the time of T cell therapy and severity of CRS did not impact the survival after alloHSCT. Conclusions: In this retrospective analysis, we found that younger age and no severe NTX to CD19 CAR therapy were associated with improved overall survival following alloHSCT. Number of relapses following alloHSCT (4 of 16 pts) appears to be lower compared to the previously reported number of relapse in the adult ALL cohort without alloHSCT (17 of 26 pts). However, the occurrence of TRM due to infection is high, likely a reflection of heavily pretreated pt population and possibly further immunosuppression from CAR + alloHSCT. The link between severe NTX and worse survival is unclear but could be related to prolonged corticosteroid use and high disease burden. While no definitive conclusion can be drawn due to a small sample size, our data suggests a certain subset of adult pts with R/R B-ALL may benefit more from alloHSCT as a consolidation therapy following CD19 CAR T cells, and acute and late infectious complications following alloHSCT should be carefully monitored. These findings should be validated prospectively and compared between different CAR constructs. Disclosures Park: Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy; Incyte: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Riviere:Fate Therapeutics: Consultancy; Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents &amp; Royalties; Juno Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding; Celgene: Consultancy.

Azacitidine (AZA) with Nivolumab (Nivo), and AZA with Nivo + Ipilimumab (Ipi) in Relapsed/Refractory Acute Myeloid Leukemia: A Non-Randomized, Prospective, Phase 2 Study

Azacitidine (AZA) with Nivolumab (Nivo), and AZA with Nivo + Ipilimumab (Ipi) in Relapsed/Refractory Acute Myeloid Leukemia: A Non-Randomized, Prospective, Phase 2 Study

AT1412, a Patient-Derived Antibody in Development for the Treatment of CD9 Positive Precursor B-Acute Lymphoblastic Leukemia

Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy, remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19 targeting immunotherapy has significantly improved patient outcomes in (relapsed) B-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. Therefore an ongoing urgency remains for the identification of additional or alternative immunotherapeutic targets for the treatment of ALL. AT1412 is an antibody that was identified from the peripheral blood memory B cell pool of a patient cured of metastatic melanoma after adoptive T-cell therapy, using a B cell immortalization technology (AIMSelect) with ectopic Bcl-6 and Bcl-xL expression as described previously [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo [manuscript submitted]. In addition to melanoma, AT1412 binds other tumor types including B-ALL, gastric, colon- and pancreatic cancer. The target of AT1412 is the tetraspanin CD9, which is expressed by more than half of all B-ALL. Expression of CD9 has been correlated with adverse prognosis [Liang et al. Cancer Biomark. 2018]. We assessed binding of this human CD9 antibody to a panel of ALL cell lines using flow cytometry. Binding of AT1412 to the B-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on the CD9 levels that we detected using a commercial CD9 antibody. AT1412 induced antibody dependent cellular cytotoxicity (ADCC) on these cells, in line with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. Importantly, these findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=30). AT1412 showed binding to 61% of B-ALL samples but not to T-ALL samples. The potential of AT1412 to induce ADCC was tested on patient samples from the same panel. Remarkably, AT1412 induced ADCC of all B-ALL samples it bound to (8 out of 14) and of none of the T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were supported by the observation that AT1412 induced B-ALL cell death when a freshly drawn whole bone marrow sample from a patient with newly diagnosed B-ALL was cocultured with AT1412. AT1412-induced cell death of B-ALL blasts occurred without affecting the monocytic, granulocytic and lymphocytic populations. This cell death was not observed when this patient's ALL blasts were incubated with AML-targeting antibodies. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the bone marrow sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human B-ALL. Taken together, the majority of precursor B-ALL blasts express CD9 and expression of CD9 is associated with a dismal outcome. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Disclosures De Jong: AIMM Therapeutics: Employment. Levie:AIMM Therapeutics: Employment. Schotte:AIMM Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties: Patent WO2017119811A1. Pos:AIMM Therapeutics: Patents &amp; Royalties: Patent WO2017119811A1. Go:AIMM Therapeutics: Employment, Patents &amp; Royalties: Patent WO2017119811A1. Yasuda:AIMM Therapeutics: Employment, Equity Ownership. Cercel:AIMM Therapeutics: Employment. van Hal-van Veen:AIMM Therapeutics: Employment. Frankin:AIMM Therapeutics: Employment. Villaudy:AIMM Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties: Patent WO2017119811A1. van Helden:AIMM Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties: Patent WO2017119811A1. van Eenennaam:AIMM Therapeutics: Employment. Spits:AIMM Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties: Patent WO2017119811A1. Hazenberg:AIMM Therapeutics: Other: Employment/equity of partner/spouse.

Targeting of Integrin α4/VCAM-1 with AVA4746 Modulates the Redox-Status and Metabolism in ALL and Angiogenesis

Background. Treatment of resistant or relapsed acute lymphoblastic leukemia (ALL) remains a challenge. Adhesion of ALL cells via integrin α4/VCAM-1 pathway to bone marrow stromal cells promotes cell adhesion-mediated drug resistance (CAM-DR). Previously, we have shown that AVA4746, a non-peptidic small molecule integrin α4 antagonist, functionally de-adheres patient-derived B-ALL cells from VCAM-1. Furthermore, we demonstrated that antagonizing VCAM-1 by AVA4746 in combination with traditional chemotherapy prolongs survival of B-ALL xenograft NSG mice derived from a relapsed B-ALL patient. The objective of the present study was to determine the underlying mechanism of the integrin α4/VCAM-1 pathway blockade using AVA4746. Method. The effect of AVA4746 intracellular levels of reactive oxygen species (ROS) in five primary B-ALL cells by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement was assessed by Seahorse XF cell mito stress test in one primary ALL using Seahorse XFp Analyzer. The effect of AVA4746 on angiogenesis was analyzed by using an endothelial tube formation assay with HUVEC cells. In vivo effect of high dose of AVA4746 (30mg/kg bid p.o.) and prolonged treatment (28 days) was assessed in a murine xenograft model of primary ALL. Results. We have previously determined that the metabolic activity was markedly decreased in two of two ALL cases by AVA4746 as assessed by WST-1 assay. Therefore, we investigated further the effect of AVA4746 on metabolism of ALL cells. For this purpose, ALL cells were treated in vitro with AVA4746 (25μM) or 0.1% DMSO as control for 2 or 4 hours. A significant increase of intracellular ROS level was consistently found in presence of AVA4746 compared to DMSO at both 2h or 4hours in all five primary B-ALL cells. According to our preliminary bioenergetics assay, after 24h treatment with AVA4746 0, 25μM, AVA4746 decreased the metabolic potential of LAX7R cells as shown by the difference in stressed OCR and stressed ECAR (P=0.0036 and 0.0056, respectively). AVA4746 significantly impeded endothelial tube formation of HUVEC cells as shown by the statistically significant differences in numbers of nodes, segments, meshes and meshes area indicating an effect of AVA4746 on angiogenesis, which will be further examined in vivo. In vivo, AVA4746 (30mg/kg bid p.o.) in combination with vincristine, dexamethasone, L-asparaginase (VDL) chemotherapy treatment prolonged survival (n=5) compared with the VDL only treated group (n=5) (MST= 87 days vs MST= 77 days; P=0.0229). There was no significant difference in survival between the PBS control group (n=4) and the AVA4746 only treatment group (n=5). Conclusion. Interrupting integrin α4/VCAM-1 pathway by AVA4746 increases intracellular ROS level of B-ALL cells and downregulates metabolic potential. AVA4746 inhibits in vitro angiogenesis. The combined treatment of high dose AVA4746 and VDL prolonged survival compared to VDL alone. As the integrin α4/VCAM-1 pathway plays a critical role in CAM-DR of B-ALL, AVA4746 may be a potential candidate for therapy of drug resistant B-ALL. Disclosures Wayne: Kite, a Gilead Company: Consultancy, Research Funding; Servier: Consultancy; AbbVie: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding.

High Mobility Group A1 Chromatin Regulators Function As Critical Drivers of Leukemogenesis in Preclinical Models of Relapsed, Pediatric B-Cell ALL

Introduction: Acute B-cell lymphoblastic leukemia (B-ALL) is the most common form of childhood leukemia and the leading cause of death in children with cancer. While therapy is often curative, about 10-15% of children will relapse with recurrent disease and abysmal outcomes. Actionable mechanisms that mediate relapse remain largely unknown. The gene encoding the High Mobility Group A1(HMGA1) chromatin regulator is overexpressed in diverse malignancies where high levels portend poor outcomes. In murine models, we discovered thatHmga1 overexpression is sufficient for clonal expansion and progression to aggressive acute lymphoid leukemia (Cancer Res 2008,68:10121, 2018,78:1890; Nature Comm 2017,8:15008). Further, HMGA1 is overexpressed in pediatric B-ALL (pB-ALL) blasts with highest levels in children who relapse early compared to those who achieve chronic remissions. Together, these findings suggest that HMGA1 is required for leukemogenesis and may foster relapse in B-ALL. We therefore sought to: 1) test the hypothesis that HMGA1 is a key epigenetic regulator required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1 in leukemogenesis. Methods: We silenced HMGA1 via lentiviral delivery of short hairpin RNAs targeting 2 different sequences in cell lines derived from relapsed pB-ALL (REH, 697). REH cells harbor the TEL-AML1 fusion; 697 cells express BCL2, BCL3, and cMYC. Next, we assessed leukemogenic phenotypes in vitro (proliferation, cell cycle progression, apoptosis, and clonogenicity) and leukemogenesis invivo. To dissect molecular mechanisms underlying HMGA1, we performed RNA-Seq and applied in silico pathway analysis. Results: There is abundant HMGA1 mRNA and protein in both pB-ALL cell lines and HMGA1 was effectively silenced by short hairpin RNA. Further, silencing HMGA1 dramatically halts proliferation in both cell lines, leading to a decrease in cells in S phase with a concurrent increase in G0/S1. Apoptosis also increased by 5-10% after HMGA1 silencing based on flow cytometry for Annexin V. In colony forming assays, silencing HMGA1 impaired clonogenicity in both pB-ALL cell lines. To assess HMGA1 function in leukemogenesis in vivo, we implanted control pB-ALL cells (transduced with control lentivirus) or those with HMGA1 silencing via tail vein injection into immunosuppressed mice (NOD/SCID/IL2 receptor γ). All mice receiving control REH cells succumbed to leukemia with a median survival of only 29 days. At the time of death, mice had marked splenomegaly along with leukemic cells circulating in the peripheral blood and infiltrating both the spleen and bone marrow. In contrast, mice injected with REH cells with HMGA1 silencing survived for &gt;40 days (P&lt;0.001) and had a significant decrease in tumor burden in the peripheral blood, spleen, and bone marrow. Similar results were obtained with 697 cells, although this model was more fulminant with control mice surviving for a median of only 17 days. To determine whether the leukemic blasts found in mice injected with ALL cells after HMGA1 silencing represented a clone that expanded because it escaped HMGA1 silencing, we assessed HMGA1 levels and found that cells capable of establishing leukemia had high HMGA1 expression, with levels similar to those observed in control cells without HMGA1 silencing. RNA-Seq analyses from REH and 697 cell lines with and without HMGA1 silencing revealed that HMGA1 up-regulates transcriptional networks involved in RAS/MAPK/ERK signaling while repressing the IDH1 metabolic gene, the latter of which functions in DNA and histone methylation. Studies are currently underway to identify effective agents to target HMGA1 pathways. Conclusions: Silencing HMGA1 dramatically disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression and epigenetic modifications. Our findings highlight the critical role for HMGA1 as a molecular switch required for leukemic transformation in pB-ALL and a rational therapeutic target that may be particularly relevant for relapsed B-ALL. Disclosures No relevant conflicts of interest to declare.