scholarly journals Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1170-1170
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Anne Kisielewski ◽  
Yang Zhang ◽  
Bruce Ruggeri ◽  
Peggy Scherle ◽  
...  
Keyword(s):  
Median Survival ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Publicly Traded ◽  
Pediatric Acute Myeloid Leukemia ◽  
Patient Derived Xenograft ◽  
Pediatric Aml ◽  
Pdx Models ◽  
Acute Myeloid

Abstract Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML. We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints. Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

scholarly journals Transcriptome Analysis Revealed the Entire Genetic Understanding of Pediatric Acute Myeloid Leukemia with a Normal Karyotype

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2850-2850
Author(s):  
Norio Shiba ◽  
Kenichi Yoshida ◽  
Yuichi Shiraishi ◽  
Shiraishi Yuichi ◽  
Yusuke Hara ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Normal Karyotype ◽  
Gene Rearrangements ◽  
Pediatric Leukemia ◽  
Pediatric Acute Myeloid Leukemia ◽  
Recurrent Mutations ◽  
Pediatric Aml ◽  
Acute Myeloid

Abstract Background: Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with a current overall survival rate of less than 70%. The pathogenesis of AML is heterogeneous and can be caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations [e.g., t(8;21), inv(16), and MLL-rearrangements] have been well established as diagnostic and prognostic markers of AML. Furthermore, recurrent mutations in FLT3, KIT, NPM1, and CEBPA have been reported in both adult and pediatric AML. Recently, massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for the prediction of the prognosis. However, these mutations are rare in pediatric AML, suggesting that other genetic alterations exist in pediatric AML. In contrast, recent reports have described NUP98-NSD1 fusion as an adverse AML prognostic marker and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harboring NUP98-NSD1 fusion. Intriguingly, PRDM16 overexpression occurs in nearly one-quarter of all children, with AML involving NUP98-NSD1-negative patients. Moreover, this overexpression is enriched in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1, and MLL-PTD). Patients and Methods: To reveal a complete registry of gene rearrangements and other genetic lesions in pediatric AML with a normal karyotype, we performed transcriptome analysis (RNA sequencing) of 61 of 70 de novo pediatric AML patients with a normal karyotype using Illumina HiSeq 2000. We could not perform RNA sequencing in nine patients because of a lack of RNA quantity or quality. Among the 70 AML patients with a normal karyotype, 33 patients overexpressed PRDM16, which was found to be strongly associated with a poor prognosis in our previous studies. All patients were enrolled and treated with AML-05 in the study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG). We also analyzed the known genetic mutations associated with these patients using the data derived from RNA sequencing. Results: A total of 144 candidate gene rearrangements, which were not observed in normal samples, were identified in 51 of 61 samples. Many of the recurrent gene rearrangements identified in this study involved previously reported targets in AML, including NUP98-NSD1, NUP98-JARID1A, CBFA2T3-GLIS2, MLL-MLLT10, and MLL-MLLT3. However, several gene rearrangements were newly identified in the current study, including MLL-SEPT6, HOXA10-HOXA-AS3, PRDM16-SKI, and CUL1-EZH2. We have also performed the validation of these novel gene rearrangements using Sanger sequencing. Most of these gene rearrangements were found in patients with a high expressionof PRDM16. In contrast, CEBPA mutations were frequently observed in patients with a low expression of PRDM16. Known gene alterations, such as FLT3-ITD and MLL-PTD, and mutations of the RAS, KIT, CEBPA, WT1, and NPM1genes were also detected using RNA sequencing. Conclusion: RNA sequencing unmasked a complexity of gene rearrangements and mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from adult counterparts, regarding the spectrum of gene rearrangements and mutations. In the present study, we identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including some novel fusion genes. These findings suggest that gene rearrangements in conjunction with mutations also play essential roles in pediatric AML. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

scholarly journals High Efficacy of Liposomal Annamycin (L-ANN) in Combination with Cytarabine in Syngeneic p53-Null AML Mouse Model

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Tomasz Zal ◽  
Rafal Zielinski ◽  
Krzysztof Grela ◽  
Roberto Cardenas-Zuniga ◽  
Stanislaw Skora ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Median Survival ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Advisory Board ◽  
Publicly Traded ◽  
Private Company ◽  
Scientific Advisory Board ◽  
Scientific Advisory ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is a heterologous hematological malignancy in which the p53-mutated subset is associated with the most guarded prognosis. Induction therapy for AML with cytarabine (cytosine arabinoside, ARA-C) is routinely used in combinations with anthracyclines. Annamycin (ANN) is an antitumoral anthracycline whose anti-leukemia activity, in contrast to doxorubicin (DOX) and daunorubicin, is unaffected by P-glycoprotein (ABCB1)-related multidrug resistance (MDR1). Unlike conventional anthracyclines, ANN accumulates in multidrug resistant cell lines, inducing DNA damage and apoptosis. Additionally, in preclinical toxicology studies, ANN displayed a greatly reduced cardiotoxicity profile compared to DOX. A liposomal formulation of ANN, termed L-Annamycin (L-ANN), is being evaluated in patients with acute myeloid leukemia (AML) in two phase Ib/IIa clinical trials in both the US and Europe. The patients are stringently followed for cardiotoxicity (Shephard et al., ASH 2020, submitted). There was no decrease in ejection fractions observed in any of the 20 patients treated to date and all cardiotoxicity biomarkers including troponin remained normal. In addition, echocardiogram analyses by the Duke cardio-oncology lab, and independently by a cardio-oncologist at Cleveland Clinic, were all normal. Considering that cardiotoxicity and MDR1-limited anthracyclines like daunorubicin are routinely used in a combination with ARA-C as induction therapy in AML, we here evaluated the combination of Ara-C with L-ANN pre-clinically. Objective: The objective of the study was to assess in vivo efficacy of the combination of L-ANN with ARA-C in a pre-clinical model of AML. Methods: The efficacy of L-ANN alone or in combination with ARA-C was investigated in a highly aggressive AML mouse model characterized by the p53-/-, MLL, ENL-FLT3, ITD mutations and genetically tagged with the cyan fluorescent protein mTurquoise2 for flow cytometry and microscopic visualization. L-ANN was intravenously administered at different dosing regimens (days 1, 2, 3 or 1, 3, 5 weekly, or 1 dose of 4 mg/kg once a week). ARA-C was administered by 5 daily intraperitoneal injections at 50 mg/kg, which was repeated every other week up to 3 times. The level of leukemia cells in peripheral circulation was analyzed by flow cytometry and AML cell presence in organ tissues was imaged by thick-mount fresh tissue confocal microscopy, in various disease stages. Results: In all tested L-ANN administration regimens (days 1, 2, 3 or 1, 3, 5 weekly, or 1 dose of 4 mg/kg once a week), we observed a significant increase in the survival of ANNARAC cohorts (combination of L-ANN with ARA-C), when compared with the respective single agents. Specifically, upon intravenous infusion of 1x105 AML1-mTurq2 cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease developed with median survival of 14 days. Administration of L-ANN on a weekly basis significantly delayed leukemia progression, as evaluated by flow cytometry and fluorescence microscopy, resulting in survival increase to 34 to 40 days, in multiple experiments. The mice treated with 50 mg/kg of ARA-C daily for 5 days a week every other week using intraperitoneal injections showed moderate to limited response to the therapy with median survival ranging from 17 to 30 days. In contrast, the median survival of animals treated with the L-ANN/ARA-C combination using different schedules ranged from 44 to 76 days, with a fraction of animals living more than 180 days after implantation of AML cells. Remarkably, imaged on day 36, the bone marrow, spleen and lungs of mice receiving combination of L-ANN (4 mg/kg once a week) with ARA-C (50 mg/kg five times per week) showed no residual disease. These results are consistent with the increased survival observed for this combination. Conclusion: This study demonstrated vastly higher efficacy of the L-ANN/ARA-C combination (ANNARAC) over that of the single agents in an immune-competent setting of an aggressive, p53-null AML model. Overall, these experiments indicate that L-ANN has the capacity to sensitize AML cells to the ARA-C induction regimen and support initiation of clinical development of L-ANN in combination with ARA-C in AML patients. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Zielinski:CNS Pharmaceuticals: Current equity holder in private company, Patents & Royalties; Moleculin Biotech, Inc.: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding. Grela:Moleculin Biotech, Inc.: Current Employment, Current equity holder in private company, Patents & Royalties. Skora:Moleculin Biotech, Inc.: Current equity holder in private company, Patents & Royalties; CNS Pharmaceuticals: Current equity holder in private company, Patents & Royalties. Fokt:CNS Pharmaceuticals: Current equity holder in private company, Patents & Royalties; Moleculin Biotech, Inc.: Consultancy, Current equity holder in private company, Patents & Royalties, Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding. Shephard:Moleculin Biotech, Inc.: Current Employment, Current equity holder in private company, Patents & Royalties. Priebe:Animal Life Sciences: Current equity holder in private company, Other: Scientific Advisor; WPD Pharmaceuticals: Current equity holder in publicly-traded company, Other: Chairman of Scientific Advisory Board, Patents & Royalties, Research Funding; CNS Pharmaceuticals: Current equity holder in private company, Other: Chairman of Scientific Advisory Board, Patents & Royalties, Research Funding; Moleculin Biotech: Current equity holder in publicly-traded company, Other: Membership of Scientific Advisory Board, Patents & Royalties, Research Funding; Reata Pharmaceuticals: Current equity holder in publicly-traded company; Houston Pharmaceuticals: Current equity holder in private company.

scholarly journals T Cell Engaging Bispecific Antibodies Produce Durable Response in Mesothelin-Positive Patient-Derived Xenograft Models of Pediatric AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1280-1280
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Colin Correnti ◽  
Kristina Pilat ◽  
Ida Lin ◽  
Albe Man Kid Chan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Median Survival ◽  
Peripheral Blood ◽  
Research Funding ◽  
Treatment Groups ◽  
Patient Derived Xenograft ◽  
Human T Cells ◽  
Xenograft Models ◽  
Pediatric Aml

Abstract Background Immunotherapy development in pediatric AML is lagging because of dearth of validated AML-specific targets. We showed recently that mesothelin (MSLN) is highly expressed on the leukemia cell surface in a subset of pediatric AML patients, and validated MSLN as a therapeutic target using antibody-drug conjugates directed against MSLN (Kaeding et al., Blood Adv, 5:2350-2361, 2021). Antibody single-chain variable region (scFv) sequences derived from amatuximab recognizing MSLN and from either blinatumomab or AMG330 targeting CD3 were used to engineer and express two MSLN/CD3-targeting BsAbs: MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG respectively. Both these antibodies demonstrated anti-leukemic activity in mice engrafted with MV4;11 cells engineered to overexpress MSLN, while they failed to show any effect in mice bearing MV4;11 cells without MSLN, confirming that these antibodies specifically targeted MSLN (Gopalakrishnapillai et al., Blood, 134:3925, 2019). Methods MSLN cell surface expression was quantitated using BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit. 3x10 6 NTPL-146 cells were injected in NSG-B2m mice and 2x10 6 DF-5 cells were injected in NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when human cells were detectable in blood. The percentage of human chimerism in mouse peripheral blood was evaluated weekly by flow cytometry. Bipsecific antibodies were administered ip at 3 mg/Kg daily for six days. Human peripheral blood pan T cells from StemCell Technologies were injected iv (3x10 6 cells per mouse) to act as effector cells. Chemotherapy (DA) consisted of 3 doses of 1.5 mg/kg daunorubicin iv and 5 doses of 50 mg/kg cytarabine ip. Mice were monitored daily and euthanized when any of the experimental endpoints were met. Results In this study, we evaluated the efficacy of two bispecific antibodies in two distinct patient-derived xenograft models of pediatric AML with endogenous MSLN expression quantitated at 6617 and 7414 MSLN antibodies bound per cell in NTPL-146 and DF-5 respectively. A Kaplan-Meier survival plot based on the time when each mouse reached experimental endpoint showed that 6/8 NTPL-146 engrafted mice receiving MSLN AMA-CD3 AMG and T cells survived disease-free until the end of the experiment at day 520 whereas all the mice in control groups had died by day 138 (Fig. 1a). The AML bone marrow load of MSLN AMA-CD3 AMG-treated mice was < 0.01% at 520 days, whereas the bone marrow load of mice from the other treatment groups was greater than 90% at the time of death, consistent with marrow failure as the proximal cause of death (Fig. 1b). These data show that treatment with MSLN AMA-CD3 AMG is curative in the vast majority of mice. Treatment with MSLN AMA-CD3 L2K and T cells increased the median survival by 109.5 days compared to untreated mice while treatment with MSLN AMA-CD3 AMG showed a complete remission in 6/8 mice (**P<0.001). Thus, the survival benefit of both MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG treated mice greatly exceeded the allogeneic effect T cells alone, which only showed a 22-day improvement in median survival. Either MSLN AMA-CD3 L2K or MSLN AMA-CD3 AMG treatment alone showed no improvement in survival compared to untreated mice. The efficacy of the potent BsAb MSLN AMA-CD3 AMG in comparison with chemotherapy (DA) was evaluated in DF-5. DA treatment, like T cell infusion, did not significantly change median survival compared to untreated mice, while BsAb MSLN AMA-CD3 AMG in the presence of human T cells was curative (Fig. 1c, **P<0.005). Mice treated with only T cells showed a rapid rise in AML cell percentage in peripheral blood until day 34 when they succumbed to disease, whereas the mice receiving T cells with BsAb had AML cell burden of less than 1% (Fig. 1d, *P<0.05). Mice treated with BsAbs showed greater expansion of allogeneic human T cells compared to mice receiving T cells alone (Fig. 1e, *P<0.05). Eight weeks post treatment initiation, the surviving mice were euthanized and AML burden in the bone marrow was evaluated. In contrast with mice receiving T cells alone, mice treated with T cells and BsAbs had no detectable AML cells (Fig. 1f, **P<0.005). Conclusion These data validate the efficacy of MSLN-targeting BsAbs in PDX models with endogenous MSLN expression. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Correnti: Link Immunotherapeutics: Current Employment. Kaeding: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

scholarly journals An Immune Checkpoint-Related Gene Signature for Predicting Survival of Pediatric Acute Myeloid Leukemia

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Feng Jiang ◽  
Xin-Yu Wang ◽  
Ming-Yan Wang ◽  
Yan Mao ◽  
Xiao-Lin Miao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Immune Checkpoint ◽  
Myeloid Leukemia ◽  
Immune Cell ◽  
Cox Regression ◽  
Secondary Data ◽  
Gene Signature ◽  
Pediatric Acute Myeloid Leukemia ◽  
Pediatric Aml ◽  
Acute Myeloid

Objective. The aim of this research was to create a new genetic signature of immune checkpoint-associated genes as a prognostic method for pediatric acute myeloid leukemia (AML). Methods. Transcriptome profiles and clinical follow-up details were obtained in Therapeutically Applicable Research to Generate Effective Treatments (TARGET), a database of pediatric tumors. Secondary data was collected from the Gene Expression Omnibus (GEO) to test the observations. In univariate Cox regression and multivariate Cox regression studies, the expression of immune checkpoint-related genes was studied. A three-mRNA signature was developed for predicting pediatric AML patient survival. Furthermore, the GEO cohort was used to confirm the reliability. A bioinformatics method was utilized to identify the diagnostic and prognostic value. Results. A three-gene (STAT1, BATF, EML4) signature was developed to identify patients into two danger categories depending on their OS. A multivariate regression study showed that the immune checkpoint-related signature (STAT1, BATF, EML4) was an independent indicator of pediatric AML. By immune cell subtypes analyses, the signature was correlated with multiple subtypes of immune cells. Conclusion. In summary, our three-gene signature can be a useful tool to predict the OS in AML patients.

Acute Differentiated Dendritic Cell Leukemia: A New Form of Pediatric Acute Myeloid Leukemia (AML) with MLL Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3263-3263
Author(s):  
Luca Lo Nigro ◽  
Laura Sainati ◽  
Anna Leszl ◽  
Elena Mirabile ◽  
Monica Spinelli ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Dendritic Cells ◽  
Dendritic Cell ◽  
Myeloid Leukemia ◽  
Fusion Transcript ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Pediatric Acute Myeloid Leukemia ◽  
Pediatric Aml ◽  
Acute Myeloid

Abstract Background: Myelomonocytic precursors from acute or chronic leukemias can differentiate to dendritic cells in vitro, but leukemias with a dendritic cell immunophenotype are rare, have been reported mainly in adults, and their molecular pathogenesis is unknown. Dendritic cells are classified as Langherans, myeloid and lymphoid/plasmacytoid cells, but leukemias arising from dendritic cells are unclassified in the FAB system. We identified a new entity of pediatric acute myeloid leukemia (AML) presenting with morphologic and immunophenotypic features of mature dendritic cells, which is characterized by MLL gene translocation. Methods and Results: Standard methods were used to characterize the morphology, immunophenotype, karyotype and MLL translocations in 3 cases of pediatric AML. The patients included two boys and one girl diagnosed with AML between 1–6 years old. Their clinical histories and findings included fever, pallor, abdominal and joint pain, adenopathy, hepatosplenomegaly, normal WBC counts but anemia and thrombocytopenia. and no evidence of CNS disease. The bone marrow aspirates were hypocellular and replaced completely by large blasts with irregular nuclei, slightly basophilic cytoplasm, and prominent cytoplasmic projections. There were no cytoplasmatic granules or phagocytosis. Myeloperoxidase and alpha napthyl esterase reactions were negative, excluding FAB M5 AML, and the morphology was not consistent with any standard FAB morphologic diagnosis. The leukemic blasts in all three cases were CD83+, CD86+, CD116+, consistent with differentiated myeloid dendritic cells, and did not express CD34, CD56 or CD117. MLL translocations were identified in all 3 cases. In the first case FISH analysis showed t(10;11)(p12;q23) and RT-PCR identified and a ‘5-MLL-AF10-3’ fusion transcript. In the second case FISH analysis showed t(9;11)(p22;q23) and RT-PCR identified and a ‘5-MLL-AF9-3’ fusion transcript. In the remaining case, the MLL gene rearrangement was identified by Southern blot analysis and RT-PCR showed an MLL-AF9 fusion transcript. The fusion transcripts in all 3 cases were in-frame. Remission induction was achieved with intensive chemotherapy, and all three patients have remained in durable remission for 30–60 months after hematopoietic stem cell transplantation. Conclusions. We have characterized a new pediatric AML entity with features of mature dendritic cells, MLL translocation and an apparently favorable prognosis. The in-frame MLL fusion transcripts suggest that chimeric MLL oncoproteins underlie its pathogenesis. The partner genes in all 3 cases were known partner genes of MLL that encode transcription factors. This study increases the spectrum of leukemias with MLL translocations. Comprehensive morphological, immunophenotypic, cytogenetic and molecular analyses are critical for this diagnosis, and will reveal its frequency and spectrum as additional cases are uncovered.

scholarly journals Distinct Genomic Landscape of Chinese Pediatric Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 38-38
Author(s):  
Ting Liu ◽  
Jianan Rao ◽  
Wenting Hu ◽  
Yuhan Liu ◽  
Huiying Sun ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Mutation Frequency ◽  
Chinese Patients ◽  
Rna Seq ◽  
Pediatric Acute Myeloid Leukemia ◽  
Genomic Landscape ◽  
Mutation Profile ◽  
Genomic Aberrations ◽  
Pediatric Aml ◽  
Acute Myeloid

Although comprehensive genomic studies have revealed key genomic aberrations in pediatric acute myeloid leukemia (AML), knowledge about Chinese patients remains lacking. Here we report the genomic landscape of Chinese pediatric AML by analyzing the sequence mutations and fusions from transcriptome sequencing (RNA-seq) of 292 cases diagnosed through 2009 to 2018 in Shanghai Children's Medical Center. Informed consents were obtained from parents for all patients. A total of 1831 non-synonymous mutations that were predicted somatic and/or associated to pediatric cancer were identified in 972 genes, including 1597 single nucleotide variants (SNV), 210 insertion/deletion (indels) and 24 internal tandem duplications (ITD), with a median of 6 mutations per case (ranging 0 to 15). Among these abnormalities, 7 aberrations occurred in more than 5% of cases in current cohort, including mutations in KIT (n=54, 18.5%), FLT3 (n=46, 15.8%), NRAS (n=28, 9.6%), CEBPA (n=23, 7.9%), ASXL2 (n=20, 6.8%), KRAS (n=16, 5.5%) and CSF3R (n=15, 5.1%). 444 potential driver variations were identified affecting 66 genes by a combined strategy of mutation pathogenicity and hotspot analysis. Each patient carried a median of one driver mutations per case (ranging 0 to 7). In addition, RNA-seq identified 227 fusions involving 99 genes in 203 out of 292 patients (69.5%), and CBL exon8/9 deletion in 12 patients (4.1%). The most prevalent fusions detected in current cohort included RUNX1-RUNX1T1 (n=82, 28.1%), KMT2A rearrangements (n=45, 15.4%) and NUP98 rearrangements (n=17, 5.8%). Furthermore, novel gene rearrangements were identified in current study, including PTPRA-FUS, ZEB2-ATIC, MSI2-UBE3C (n=1 each). Distinct genomic aberration profile was revealed while comparing our results to the mutation profile characterized in Children's Oncology Group (COG)-National Cancer Institute (NCI) TARGET AML initiative representing the Western pediatric AML cohort. A total of 16 recurrently mutated genes were identified with significantly (two-sided fisher exact test) different mutation frequency. Among these, 7 genes mutated more frequently in Chinese patients, including KIT (18.5% vs 12.8% in Chinese and Western cohort, respectively. p=0.027), ASXL2 (6.8% vs 3.6%, p=0.043), CSF3R (5.1% vs 2.4%, p=0.044), JAK2 (3.4% vs 0.0%, p<0.001), DNM2 (2.7% vs 0.0%, p<0.001), KDM6A (2.1% vs 0.0%, p<0.001) and KMT2C (1.7% vs 0.0%, p=0.003). On the other hand, mutations in FLT3 (15.8% vs 33.0%, p<0.001), NRAS (9.6 vs 30.9%, p<0.001), KRAS (5.5% vs 12.8%, p<0.001), WT1 (2.4% vs 13.6%, p<0.001), NPM1 (2.4% vs 10.3%, p<0.001), PTPN11 (3.8% vs 8.1%, p=0.016), TET2 (1.0% vs 5.2%, p=0.001), CBL sequence mutation (0.0% vs 3.0%, p<0.001) and IKZF1 (0.3% vs 2.7%, p=0.018) were occurred less frequently in Chinese patients. Notably, the RAS signaling pathway as a whole was significantly less frequently mutated in Chinese patients (35.6% vs 71.0%, p<0.001). Furthermore, distinct associations between mutations and FAB subtypes were also observed. For example, NF1 mutations were significantly enriched with subtype M5 in Chinese patients (p=0.003), which was previously reported as co-mutated with CBFB-MYH11 fusion with associated with subtype M4. Survival analysis revealed key genomic aberrations associated with patient prognosis. Variants significantly (log-rank test) associated with better event free survival rate included mutations in CEBPA (p=0.023), NPM1 (p=0.026) and GATA2 (p=0.016). On the other hand, CBFA2T3-GLIS2 (p=0.028), nucleoporin gene family related fusions (including NUP98, NUP214 and NUP153, p<0.001), FUS related fusions (p=0.030), mutations in RUNX1 (p<0.001) and FLT3 (p=0.003) were associated with worse prognosis. A revised risk stratification model was proposed based on these associations observed. Characterized a first comprehensive genomic landscape of Chinese pediatric AML, our results reveal a distinct mutation profile as compared to the Western cohort, in terms of both mutation frequency and patterns of mutation co-occurrence. These findings further reveal the complexity of pediatric AML and highlight the importance of tailored risk stratification for Chinese patients in clinical management. Disclosures No relevant conflicts of interest to declare.

scholarly journals Imetelstat Significantly Reduces Leukemia Stem Cells in Patient-Derived Xenograft Models of Pediatric AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3352-3352
Author(s):  
Sonali P. Barwe ◽  
Fei Huang ◽  
E. Anders Kolb ◽  
Anilkumar Gopalakrishnapillai
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Research Funding ◽  
Annexin V ◽  
Telomerase Activity ◽  
Leukemia Stem Cells ◽  
Patient Derived Xenograft ◽  
Pediatric Aml ◽  
Pdx Models

Abstract Introduction Acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite the use of maximally intensive therapy, 20% of patients experience recurrent disease. These patients are also burdened with significant treatment-related toxicities. To improve survival in pediatric AML, novel targeted therapies that are more effective and less toxic are needed. Telomerase inhibition has been shown to be effective in reducing leukemic burden and eradicating leukemia stem cells (LSCs) in syngeneic mouse models of AML and in patient-derived xenograft (PDX) models of adult AML (Bruedigam et al., 2014). Recent transcriptome analyses demonstrate that the genomic landscape of pediatric AML is distinct from adult AML (Bolouri et al., 2018). In fact, mutations in the telomerase complex components are infrequent in pediatric AML unlike adult AML patients (Aalbers et al., 2013). However, similar to what is seen in adult patients, Aalbers et al. identified that telomere lengths in pediatric AML cells were shortened compared to normal leukocytes, and pediatric AML patients with the shortest telomere length tend to have shorter overall survival. Furthermore, the 5-year survival rate was 88% for pediatric AML patients who had lower telomerase activity, and 43% for those patients with higher telomerase activity, suggesting telomerase activity could be an important prognostic factor in pediatric AML patients (Verstovsek et al., 2003). Imetelstat is an oligonucleotide that specifically binds with high affinity to the RNA template of telomerase and is a potent, competitive inhibitor of telomerase enzymatic activity (Asai et al., 2003; Herbert et al., 2005). In this study, we evaluated if imetelstat has anti-leukemia activity in pediatric AML PDX models. Results The PDX lines tested in this study were derived using samples from pediatric AML patients who were 1-14 years old, representing different FAB subtypes. Mouse passaged pediatric AML PDX lines (n=6) were treated ex vivo with imetelstat or mismatch oligo control and the viability of LSC (CD34+CD38low population) was determined at 48 or 96 h by staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Imetelstat treatment significantly increased apoptosis/death (PI+/annexin V+) of the LSC population in a dose-dependent manner in all PDX lines evaluated (Fig. 1A, B), while it had limited activity on LSCs in normal pediatric bone marrow samples (n=4). The efficacy of imetelstat either alone or in combination with chemotherapy or azacitidine was evaluated in two distinct PDX models of pediatric AML in vivo. Mice engrafted with both NTPL-377 and DF-2 lived longer when treated with imetelstat than the untreated mice (Fig. 1C, D, n=5 each, P<0.05). Mice receiving standard chemotherapy consisting of cytarabine and daunorubicin or azacitidine showed prolonged survival compared to the untreated mice. Interestingly, sequential administration of imetelstat following chemotherapy treatment provided additional benefit over chemotherapy alone (P<0.01). Concurrent treatment of azacitidine and imetelstat further extended survival of these mice compared to azacitidine alone (P<0.05). At the end of the in vivo studies, the percentage of LSC population was evaluated in the bone marrow of mice post euthanasia. There was a significant reduction of LSC population in mice treated with imetelstat compared to those treated with the mismatch oligo (Fig. 1E, F, P<0.05). Neither chemotherapy nor azacitidine alone affected LSC population compared to untreated mice. However, imetelstat significantly reduced the LSC population when combined with chemotherapy or azacitidine compared to single agent (P<0.05). These results were confirmed by secondary transplantation in mice, which showed delayed engraftment of cells isolated from imetelstat treated mice (Fig. 1G, H). Conclusions Imetelstat treatment of pediatric AML PDX samples showed significant dose- and time-dependent effects on the viability of the LSCs to induce cell apoptosis/death. These results were corroborated in vivo in two distinct PDX models which showed reduced LSC population and increased median survival in mice with imetelstat treatment. Combining imetelstat with chemotherapy or azacitidine further enhanced activity against LSCs, suggesting imetelstat could represent an effective therapeutic strategy for pediatric AML. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Huang: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Gopalakrishnapillai: Geron: Research Funding.

scholarly journals Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia in First Complete Remission after 5-Azacitidine and Venetoclax: A Multicenter Retrospective Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3962-3962
Author(s):  
Oren Pasvolsky ◽  
Shai Shimony ◽  
Ron Ram ◽  
Avichai Shimoni ◽  
Liat Shargian-Alon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Median Survival ◽  
Cumulative Incidence ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hematopoietic Cell ◽  
Remission Induction ◽  
Intensive Chemotherapy ◽  
Intensive Group ◽  
Acute Myeloid

Abstract The therapeutic landscape for acute myeloid leukemia (AML) has evolved in recent years with the introduction of hypomethylating agents (HMA) and venetoclax in patients previously deemed unfit for curative - intent treatment. Some of these patients undergo allogeneic hematopoietic cell transplant (alloHCT), yet there are scarce data regarding transplantation outcomes. We conducted a multicenter nationwide retrospective cohort study to evaluate outcomes of patients with AML who underwent alloHCT in first CR (CR1) after frontline treatment with 5-azacitidine plus venetoclax (aza-ven group). In addition, we collected a historical control group of patients who achieved CR1 following first line intensive chemotherapy followed by alloHCT (intensive group). 24 patients in the aza-ven group were transplanted between 2019 and 2021. Compared to the intensive group, patients in the aza-ven group were older (median age 71.7 vs. 58.4 years, p <0.001), had higher incidence of therapy related AML and AML with antecedent hematologic disorder (p <0.001) and had more often adverse cytogenetics (p=0.022). They had a higher percentage of allografts from matched unrelated donors, and reduced intensity conditioning was more commonly used (Table 1). Median follow up was 8 (range, 0 to 25) months in the aza-ven group and 23 (range, 4 to 56) months in the intensive group. Estimated 12 months non relapse mortality was 19.1% in the aza-ven group and 11.8% in the intensive group (p=0.492). The estimated median relapse free survival (RFS) was not reached in the aza-ven group and was 19.3 months (CI 95% 1-38) in the intensive group. There was no difference between the two groups in 12 months RFS (58% and 54% in the aza-ven group and intensive group, respectively, p = 0.892). The estimated median survival of the aza-ven group was not reached and the 12 months overall survival (OS) rate was 63.2%. The estimated median survival of the intensive group was 50 months (CI 95% 5 - 96) and the 12 months OS rate was 70.8%. There was no statistical differences between the two groups regarding OS (p = 0.58). In a subgroup Cox regression analysis of the aza-ven group, adverse ELN 2017 risk category and HCT-CI score ≥3 were predictive of decreased RFS, both in univariate analysis (UVA) and in multivariate analysis (MVA) (HR 10.56, CI 95%1.64-68.1, p=0.013 and HR 6.43, CR 95% 1.34-30.75, p=0,02, respectively). Graft source (alternative vs. matched donor) and HCT-CI score ≥3 were predictive of decreased OS in UVA (HR 19.45, CI 95% 1.66-228.13, p= 0.018 and HR 5.93, CI 95% 1.13-31.05, p=0.03], yet in MVA neither of these factors retained their predictive value. The cumulative incidence of acute GVHD at 6 months was similar between groups: 58% in the aza-ven group vs. 62% in the intensive group (p=0.39). Likewise, there was no difference in the cumulative incidence of chronic GVHD at 12 months: 40% vs 42%, respectively (p=0.747) In conclusion, our data suggests that alloHCT for AML patients achieving first CR with aza-ven appears feasible, with short term post-transplant outcomes comparable to those expected after traditional intensive chemotherapy. Our results were collected in the real world setting, and patients in the aza-ven group were older and had inherently worse leukemia characteristics, including more secondary AML and more adverse cytogenetic features. Future research is warranted to decipher the true spectrum of AML patients who could benefit from remission induction with this less intensive regimen prior to alloHCT. Figure 1 Figure 1. Disclosures Ram: Gilead: Honoraria; Novartis: Honoraria. Wolach: Janssen: Consultancy; Abbvie: Consultancy, Honoraria, Research Funding; Astellas: Consultancy; Amgen: Research Funding; Novartis: Consultancy; Neopharm: Consultancy. Yeshurun: Astellas: Consultancy; Janssen: Consultancy.

scholarly journals Preliminary Results from a Phase 1 Study of APVO436, a Novel Anti-CD123 x Anti-CD3 Bispecific Molecule, in Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Justin M. Watts ◽  
Tara Lin ◽  
Eunice S. Wang ◽  
Alice S. Mims ◽  
Elizabeth H. Cull ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Phase 1 ◽  
Cytokine Release ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Acute Myeloid

Introduction Immunotherapy offers the promise of a new paradigm for patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). CD123, the IL-3 receptor alpha-chain, represents an attractive target for antibody therapies because of its high expression on AML/MDS blasts and leukemic stem cells compared to normal hematopoietic stem and progenitor cells. APVO436, a novel bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule, depleted CD123+ cells in AML patient samples ex vivo (Godwin et al. ASH 2017), reduced leukemia engraftment in a systemic AML xenograft model (Comeau et al. AACR 2018), and transiently reduced peripheral CD123+ cells in non-human primates with minimal cytokine release and in a dose-dependent fashion (Comeau et al. AACR 2019). These data provide a basis for the clinical application of APVO436 as a treatment in AML and MDS. Here, we report preliminary data from a first-in-human dose-escalation study of APVO436 in patients with R/R AML and high-risk MDS. Study Design/Methods This ongoing Phase 1/1b study (ClinicalTrials.gov: NCT03647800) was initiated to determine the safety, immunogenicity, pharmacokinetics, pharmacodynamics, and clinical activity of APVO436 as a single agent. Major inclusion criteria were: R/R AML with no other standard treatment option available, R/R MDS with > 5% marrow blasts or any peripheral blasts and failure of a hypomethylating agent, ECOG performance status ≤ 2, life expectancy > 2 months, white blood cells ≤ 25,000 cells/mm3, creatinine ≤ 2 x upper limit of normal (ULN), INR and PTT < 1.5 x ULN and alanine aminotransferase < 3 x ULN. Patients were not restricted from treatment due to cytogenetic or mutational status. Intravenous doses of APVO436 were administered weekly for up to six 28-day cycles (24 doses) with the option to continue dosing for up to 36 total cycles (144 doses). Flat and step dosing regimens were escalated using a safety-driven modified 3 + 3 design. Pre-medication with diphenhydramine, acetaminophen, and dexamethasone was administered starting with dose 1 to mitigate infusion related reactions (IRR) and cytokine release syndrome (CRS). First doses and increasing step doses of APVO436 were infused over 20-24 hours followed by an observation period of 24 hours or more. Bone marrow biopsies were performed every other cycle with responses assessed by European Leukemia Net 2017 criteria for AML or International Working Group (IWG) 2006 criteria for MDS. Results The data cut-off for this interim analysis was July 9, 2020. Twenty-eight patients with primary R/R AML (n=19), therapy-related R/R AML (n=3), or high-risk MDS (n=6) have been enrolled and received a cumulative total of 186 doses. The number of doses received per patient ranged from 1 to 43 (mean of 6.4 doses). Most patients discontinued treatment due to progressive disease; however, blast reduction was achieved in 2 patients, with one patient with MDS maintaining a durable response for 11 cycles before progressing. APVO436 was tolerated across all dose regimens in all cohorts tested. The most common adverse events (AEs), regardless of causality, were edema (32%), diarrhea (29%), febrile neutropenia (29%), fever (25%), hypokalemia (25%), IRR (21%), CRS (18%), chills (18%), and fatigue (18%). AEs ≥ Grade 3 occurring in more than one patient were: febrile neutropenia (25%), anemia (18%), hyperglycemia (14%), decreased platelet count (11%), CRS (11%), IRR (7%), and hypertension (7%). After observing a single dose limiting toxicity (DLT) at a flat dose of 9 µg, step dosing was implemented and no DLTs have been observed thereafter. No treatment-related anti-drug antibodies (ADA) were observed. Transient serum cytokine elevations occurred after several reported IRR and CRS events, with IL-6 most consistently elevated. Conclusions Preliminary results indicate that APVO436 is tolerated in patients with R/R AML and MDS at the doses and schedules tested to date, with a manageable safety profile. Dose escalation continues and the results will be updated for this ongoing study. Disclosures Watts: BMS: Membership on an entity's Board of Directors or advisory committees; Aptevo Therapeutics: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees. Lin:Ono Pharmaceutical: Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bio-Path Holdings: Research Funding; Astellas Pharma: Research Funding; Aptevo: Research Funding; Celgene: Research Funding; Genetech-Roche: Research Funding; Celyad: Research Funding; Prescient Therapeutics: Research Funding; Seattle Genetics: Research Funding; Mateon Therapeutics: Research Funding; Jazz: Research Funding; Incyte: Research Funding; Gilead Sciences: Research Funding; Trovagene: Research Funding; Tolero Pharmaceuticals: Research Funding. Wang:Abbvie: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy; Jazz Pharmaceuticals: Consultancy; Bristol Meyers Squibb (Celgene): Consultancy; PTC Therapeutics: Consultancy; Stemline: Speakers Bureau; Genentech: Consultancy; Pfizer: Speakers Bureau. Mims:Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Agios: Consultancy; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board; Abbvie: Membership on an entity's Board of Directors or advisory committees. Cull:Aptevo Therapeutics: Research Funding. Patel:Agios: Consultancy; Celgene: Consultancy, Speakers Bureau; DAVA Pharmaceuticals: Honoraria; France Foundation: Honoraria. Shami:Aptevo Therapeutics: Research Funding. Walter:Aptevo Therapeutics: Research Funding. Cogle:Aptevo Therapeutics: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Chenault:Aptevo Therapeutics: Current Employment, Current equity holder in publicly-traded company. Macpherson:Aptevo Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chunyk:Aptevo Therapeutics: Current Employment, Current equity holder in publicly-traded company. McMahan:Aptevo Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gross:Aptevo Therapeutics: Current Employment, Current equity holder in publicly-traded company. Stromatt:Aptevo Therapeutics: Current equity holder in publicly-traded company.

scholarly journals In Vivo Evaluation of Mesothelin As a Therapeutic Target in Pediatric Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1370-1370 ◽  
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Allison Kaeding ◽  
Christoph Schatz ◽  
Anette Sommer ◽  
Soheil Meshinchi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Combination Therapy ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Antibody Drug Conjugate ◽  
Pediatric Acute Myeloid Leukemia ◽  
Pediatric Aml ◽  
Acute Myeloid ◽  
Antibody Drug

Pediatric acute myeloid leukemia (AML) continues to have a cure rate of only 50% despite the use of highly intensive cytotoxic chemotherapy. Transcriptome sequencing of several AML samples by the NCI/COG TARGET AML Initiative identified mesothelin (MSLN) to be highly overexpressed in about one-third of pediatric AML (Tarlock et al., Blood, 128:2873, 2016). Because MSLN is not expressed in normal bone marrow samples (Fan et al., Blood, 130:3792, 2017) and only to a low level in other human organs and tissues, MSLN is an attractive therapeutic target for pediatric AML (Kaeding et al., Blood, 130:2641, 2017). The anti-MSLN antibody-drug conjugate (ADC) anetumab ravtansine (BAY 94-9343) generated by conjugating MSLN-antibody with tubulin inhibitor DM4 (Meso-ADC), and isotype control antibody conjugated with the same drug (Iso-ADC) were used to evaluate the efficacy of MSLN targeting in vivo. MSLN-overexpressing K562 (K562-MSLN) CML cells and MV4;11 (MV4;11-MSLN) AML cells were generated by lentiviral transduction of MSLN cDNA. Cell line-derived xenografts (CDX) were created by injecting the MSLN-transduced or parental (MSLN-) cells into NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when the median percentage of human cells in mouse peripheral blood was greater than 0.5%. K562-MSLN CDX mice treated with Meso-ADC (5 mg/Kg Q3dx3, i.v.) survived a median of 46 days longer than those treated with Iso-ADC (P=0.0011) and significantly longer than comparison groups, including K562-MSLN CDX mice treated with daunorubicin and Ara-C (DA, P=0.0008) or untreated (P=0.0018) (Fig. 1A). Median survival of K562 CDX mice treated with Meso-ADC, Iso-ADC, or untreated was similar (Fig. 1B). MV4;11-MSLN CDX mice treated with Meso-ADC exhibited complete remission and remained disease-free at 1 year post cell injection, with AML cell burden remaining <0.1% throughout the study period (Fig. 1C). In contrast, MV;11-MSLN CDX mice treated with Iso-ADC or untreated succumbed to disease at 72 and 38 days, respectively. Taken together, these results indicate that Meso-ADC was efficacious in reducing leukemia burden, and this effect required MSLN expression in target cells. We have generated a panel of patient-derived xenograft (PDX) lines by transplanting and serially propagating primary pediatric AML samples into NSG-SGM3 mice. The efficacy of Meso-ADC was also evaluated in a systemic PDX model using a MSLN+ PDX line (NTPL-146). NTPL-146 PDX mice treated with Meso-ADC (5 mg/Kg, Q3dx3 -x2 cycles) survived a median of 50 days longer than those treated with Iso-ADC (P=0.0018, Fig. 1D, arrows indicate time when each treatment cycle was initiated). In an independent experiment with NTPL-146 PDX mice, a survival benefit of Meso-ADC treatment over no treatment was observed after 1 cycle of Meso-ADC treatment (5 mg/Kg, Q3dx3, P=0.0019, Fig. 1E). Additionally, a combination therapy strategy with daunorubicin and Ara-C followed by Meso-ADC (DA -> Meso-ADC) resulted in improved median survival over Meso-ADC (P=0.0027) or DA treatment alone (P=0.0018) (Fig. 1E). The disseminated MSLN+ leukemia mouse models described herein support MSLN-targeted antibody-drug conjugate as a potential treatment strategy in MSLN+ AML. Furthermore, we provide the first in vivo demonstration of synergy between MSLN-targeted therapy and conventional chemotherapy in MSLN+ AML, warranting additional investigation to validate and optimize novel strategies for combination therapy. Figure 1 Disclosures Kaeding: Celgene: Employment. Schatz:Bayer AG: Employment. Sommer:Bayer AG: Employment, Equity Ownership.

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