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In an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN–DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

Modern targeted cancer therapies rely on the overexpression of tumor associated antigens with very little to no expression in normal cell types. Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein that has been identified in many different tumor types, including lung adenocarcinomas, ovarian carcinomas, and most recently in hematological malignancies, including acute myeloid leukemia (AML). Although the function of mesothelin is widely unknown, interactions with MUC16/CA125 indicate that mesothelin plays a role in the regulation of proliferation, growth, and adhesion signaling. Most research on mesothelin currently focuses on utilizing mesothelin to design targeted cancer therapies such as monoclonal antibodies, antibody–drug conjugates, chimeric antigen receptor T and NK cells, bispecific T cell engaging molecules, and targeted alpha therapies, amongst others. Both in vitro and in vivo studies using different immunotherapeutic modalities in mesothelin-positive AML models highlight the potential impact of this approach as a unique opportunity to treat hard-to-cure AML.

Children with acute myeloid leukemia (AML) have a poor prognosis despite the intensification of chemotherapy. Future efforts to improve outcomes should focus on more precise targeting of leukemia cells. CD123, or IL3RA, is expressed on the surface of nearly all pediatric AML samples and is a high-priority target for immunotherapy. The efficacy of an investigational dual-affinity retargeting antibody (DART) molecule (CD123 × CD3; MGD006 or flotetuzumab) was assessed in two distinct patient-derived xenograft (PDX) models of pediatric AML. MGD006 simultaneously binds to CD123 on target cells and CD3 on effector T cells, thereby activating T cells and redirecting them to induce cytotoxicity in target cells. The concurrent treatment of cytarabine and MGD006 was performed to determine the effect of cytarabine on T-cell counts and MGD006 activity. Treatment with MGD006 along with an allogeneic human T-cell infusion to act as effector cells induced durable responses in both PDX models, with CD123 positivity. This effect was sustained in mice treated with a combination of MGD006 and cytarabine in the presence of T cells. MGD006 enhanced T-cell proliferation and decreased the burden of AML blasts in the peripheral blood with or without cytarabine treatment. These data demonstrate the efficacy of MGD006 in prolonging survival in pediatric AML PDX models in the presence of effector T cells and show that the inclusion of cytarabine in the treatment regimen does not interfere with MGD006 activity.

Simple Summary Immunotherapy development in pediatric AML has been slow due to the paucity of validated AML-specific targets. We recently identified mesothelin (MSLN) as a therapeutic target in pediatric AML. Mice receiving T cell engaging bispecific antibodies (BsAbs) targeting MSLN and CD3 achieved complete remission and durable responses in two MSLN-positive patient-derived xenograft (PDX) models. This is a first report showing MSLN-targeting BsAbs are a viable immunotherapy for MSLN-positive pediatric AML. Abstract Advances in the treatment of pediatric AML have been modest over the past four decades. Despite maximally intensive therapy, approximately 40% of patients will relapse. Novel targeted therapies are needed to improve outcomes. We identified mesothelin (MSLN), a well-validated target overexpressed in some adult malignancies, to be highly expressed on the leukemic cell surface in a subset of pediatric AML patients. The lack of expression on normal bone marrow cells makes MSLN a viable target for immunotherapies such as T-cell engaging bispecific antibodies (BsAbs) that combine two distinct antibody-variable regions into a single molecule targeting a cancer-specific antigen and the T-cell co-receptor CD3. Using antibody single-chain variable region (scFv) sequences derived from amatuximab-recognizing MSLN, and from either blinatumomab or AMG330 targeting CD3, we engineered and expressed two MSLN/CD3-targeting BsAbs: MSLNAMA-CD3L2K and MSLNAMA-CD3AMG, respectively. Both BsAbs promoted T-cell activation and reduced leukemic burden in MV4;11:MSLN xenografted mice, but not in those transplanted with MSLN-negative parental MV4;11 cells. MSLNAMA-CD3AMG induced complete remission in NTPL-146 and DF-5 patient-derived xenograft models. These data validate the in vivo efficacy and specificity of MSLN-targeting BsAbs. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients.

In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.

Induced pluripotent stem cells (iPSCs) provide an extraordinary tool for disease modeling owing to their potential to differentiate into the desired cell type. The differentiation of iPSCs is typically performed on 2-dimensional monolayers of stromal cell or animal tissue derived extracellular matrices. Recent advancements in disease modeling have utilized iPSCs in 3-dimensional (3D) cultures to study diseases such as muscular dystrophy, cardiomyopathy, and pulmonary fibrosis. However, these approaches are yet to be explored in modeling the hematological malignancies. Transient myeloproliferative disorder (TMD) is a preleukemic stage, which is induced in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. In this study, we established a synthetic 3D iPSC culture system for modeling TMD via hematopoietic differentiation of customized iPSCs. A chemically cross-linkable PEG hydrogel decorated with integrin binding peptide was found to be permissive of hematopoietic differentiation of iPSCs. It provided a cost-effective system for the generation of hematopoietic stem and progenitor cells (HSPCs) with higher yield of early HSPCs compared to traditional 2D culture on Matrigel coated dishes. Characterization of the HSPCs produced from the iPSC lines cultured in 3D showed that the erythroid population was reduced whereas the megakaryoid and myeloid populations were significantly increased in GATA1 mutant trisomic line compared to disomic or trisomic lines with wild-type GATA1, consistent with TMD characteristics. In conclusion, we have identified a cost-effective tunable 3D hydrogel system to model TMD.

Translocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion-driven leukemia is sensitive to the menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of proleukemogenic transcription factors such as Meis1 in NUP98-fusion-transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for the maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion-driven AML to test the in vivo efficacy of menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that menin-MLL1 inhibition simultaneously suppresses a proleukemogenic gene expression program, including downregulation of the HOXa cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

Relapse remains a major obstacle to achieving 100% overall survival rate in pediatric hematologic malignancies like acute lymphoblastic leukemia (ALL). Relapse often results from the development of chemoresistance. One of the mechanisms of chemoresistance involves ALL cell interactions with the bone marrow (BM) microenvironment, providing a sanctuary. This phenomenon is known as BM microenvironment-induced chemoprotection. Members of the transmembrane 4 superfamily (tetraspanins; TSPANs) are known to mediate microenvironmental interactions and have been extensively studied in solid tumors. Although the TSPAN family member CD81 is a minimal residual disease marker, its biological role in ALL is not well characterized. We show for the first time that CD81 knockout induces chemosensitivity, reduces cellular adhesion, and disrupts in vivo BM homing and engraftment in B-ALL. This chemosensitization is mediated through control of Bruton tyrosine kinase signaling and induction of p53-mediated cell death. We then show how CD81-related signaling can be disrupted by treatment with the epigenetic drug combination of DNA hypomethylating agent azacitidine (aza) and histone deacetylase inhibitor panobinostat (pano), which we previously used to sensitize ALL cells to chemotherapy under conditions that promote BM microenvironment-induced chemoprotection. Aza/pano-mediated modulation of CD81 surface expression is involved in decreasing BM load by promoting ALL cell mobilization from BM to peripheral blood and increasing response to chemotherapy in disseminated patient-derived xenograft models. This study identifies the novel role of CD81 in BM microenvironment-induced chemoprotection and delineates the mechanism by which aza/pano successfully sensitizes ALL cells via modulation of CD81.

The development of resistance to anti-cancer therapeutics remains one of the core issues preventing the improvement of survival rates in cancer. Therapy resistance can arise in a multitude of ways, including the accumulation of epigenetic alterations in cancer cells. By remodeling DNA methylation patterns or modifying histone proteins during oncogenesis, cancer cells reorient their epigenomic landscapes in order to aggressively resist anti-cancer therapy. To combat these chemoresistant effects, epigenetic modifiers such as DNA hypomethylating agents, histone deacetylase inhibitors, histone demethylase inhibitors, along with others have been used. While these modifiers have achieved moderate success when used either alone or in combination with one another, the most positive outcomes were achieved when they were used in conjunction with conventional anti-cancer therapies. Epigenome modifying drugs have succeeded in sensitizing cancer cells to anti-cancer therapy via a variety of mechanisms: disrupting pro-survival/anti-apoptotic signaling, restoring cell cycle control and preventing DNA damage repair, suppressing immune system evasion, regulating altered metabolism, disengaging pro-survival microenvironmental interactions and increasing protein expression for targeted therapies. In this review, we explore different mechanisms by which epigenetic modifiers induce sensitivity to anti-cancer therapies and encourage the further identification of the specific genes involved with sensitization to facilitate development of clinical trials.

Approximately 1%–2% of children with Down syndrome (DS) develop acute myeloid leukemia (AML) prior to age 5 years. AML in DS children (ML-DS) is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated short form of GATA1 (GATA1s). Trisomy 21 and GATA1s together are sufficient to induce transient abnormal myelopoiesis (TAM) exhibiting pre-leukemic characteristics. Approximately 30% of these cases progress into ML-DS by acquisition of additional somatic mutations. We employed disease modeling in vitro by the use of customizable induced pluripotent stem cells (iPSCs) to generate a TAM model. Isogenic iPSC lines derived from the fibroblasts of DS individuals with trisomy 21 and with disomy 21 were used. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system was used to introduce GATA1 mutation in disomic and trisomic iPSC lines. The hematopoietic stem and progenitor cells (HSPCs) derived from GATA1 mutant iPSC lines expressed GATA1s. The expression of GATA1s concomitant with loss of full-length GATA1 reduced the erythroid population, whereas it augmented megakaryoid and myeloid populations, characteristic of TAM. In conclusion, we have developed a model system representing TAM, which can be used for modeling ML-DS by stepwise introduction of additional mutations., Graphical Abstract, Acute myeloid leukemia in Down syndrome children is characterized by the mutation in GATA1, resulting in N-terminally truncated mutant GATA1 (GATA1s). Trisomy 21 and GATA1s together induce transient abnormal myelopoiesis (TAM) exhibiting pre-leukemic characteristics. The expression of GATA1s reduced the erythroid population and augmented megakaryoid and myeloid populations, characteristic of TAM.

Background Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. Methods Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4–11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. Results Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. Conclusions Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

Acute leukemia represents the most common pediatric malignancy comprising diverse subtypes with varying prognosis and treatment outcomes. New and targeted treatment options are warranted for this disease. Patient-derived xenograft (PDX) models are increasingly being used for preclinical testing of novel treatment modalities. A novel approach involving targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was employed to compare 25 primary pediatric acute leukemia samples and their corresponding PDX samples. A comparison of the primary samples and PDX samples revealed a high concordance between single nucleotide variants and gene fusions whereas other complex structural variants were not as consistent. The presence of gene fusions representing the major driver mutations at similar allelic frequencies in PDX samples compared to primary samples and over multiple passages confirms the utility of PDX models for preclinical drug testing. Characterization and tracking of these novel cryptic fusions and exonal variants in PDX models is critical in assessing response to potential new therapies.

Acute myeloid leukemia (AML) in children remains deadly, despite the use of maximally intensive therapy. Because leukemia stem cells (LSCs) significantly contribute to chemoresistance and relapse, therapies that specifically target the LSCs are likely to be more beneficial in improving outcome. LSCs are known to have high telomerase activity and telomerase activity is negatively correlated with survival in pediatric AML. We evaluated the preclinical efficacy of imetelstat, an oligonucleotide inhibitor of telomerase activity in patient-derived xenograft (PDX) lines of pediatric AML. Imetelstat treatment significantly increased apoptosis/death of the LSC population in a dose-dependent manner in six pediatric AML PDX lines ex vivo, while it had limited activity on the stem cell population in normal bone marrow specimens. These results were validated in vivo in two distinct PDX models wherein imetelstat as single agent or in combination with chemotherapy greatly reduced the LSC percentage and prolonged median survival. Imetelstat combination with DNA hypomethylating agent azacitidine was also beneficial in extending survival. Secondary transplantation experiments showed delayed engraftment and improved survival of mice receiving imetelstat-treated cells, confirming the diminished LSC population. Thus, our data suggest that imetelstat represents an effective therapeutic strategy for pediatric AML.

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 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 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.

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 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.

Background Children with Down syndrome (DS) have a high risk for acute myeloid leukemia (DS-ML). Genomic characterization of DS-ML blasts showed the presence of unique mutations in GATA1, an essential hematopoietic transcription factor, leading to the production of a truncated from of GATA1 (GATA1s). GATA1s together with trisomy 21 is sufficient to develop a pre-leukemic condition called transient abnormal myelopoiesis (TAM). Approximately thirty percent of these cases progress into DS-ML by acquisition of additional somatic mutations in a step-wise manner. We previously developed a model for TAM by introducing disease-specific GATA1 mutation in trisomy 21 induced pluripotent stem cells (iPSCs) leading to the production of N-terminally truncated short form of GATA1 (GATA1s) (Barwe et al., 2021). In this study, we introduced co-operating mutation in STAG2, a member of the cohesin complex recurrently mutated in DS-ML but not in TAM, and evaluated its effect on hematopoietic differentiation. Methods Two different iPSC lines with trisomy 21 with or without GATA1 mutation as described in Barwe et al., 2021, were used. CRISPR/Cas9 gene editing was performed to introduce STAG2 mutation to generate a knockout of STAG2. Hematopoietic differentiation of these iPSC lines was performed using STEMdiff Differentiation kit. ProteinSimple Wes system was used for western blot analysis. Multi-dimensional flow cytometry was used for immunophenotypic analysis of megakaryoblasts cultured in lineage expansion media for 5 days. Multi-lineage colony forming potential was assessed by Methocult colony forming assay using day 10 hematopoietic stem progenitor cells (HSPCs). Results Hematopoietic differentiation of GATA1 and STAG2 double mutants in two independent trisomy 21 iPSC lines confirmed GATA1s expression and the loss of functional STAG2 protein (Fig. 1A). GATA1s expressing HSPCs collected on day 12 post differentiation showed reduced erythroid (CD71+CD235+) and increased megakaryoid (CD34+CD41+ within CD41+ compartment) and myeloid (CD18+CD45+) population compared to disomy 21 HSPCs with wild-type GATA1, consistent with our previous study (Fig. 2B). STAG2 knockout HSPCs showed higher erythroid population (P=0.033 and 0.016 in T21-1S and T21-2S respectively) and reduced myeloid population while it had no significant effect on the megakaryoid population in both iPSC lines. The GATA1s/STAG2 knockout HSPCs showed reduced erythroid, but higher megakaryoid and myeloid population compared to wild-type HSPCs. Strikingly, the immature megakaryoid population was significantly higher in the double mutant HSPCs compared to single mutant alone in both iPSC lines (P=0.005 and 0.004 for T21-1GS and T21-2GS respectively), indicating that the STAG2knockout co-operated with GATA1s for increasing megakaryoid population. The trisomy 21 iPSC line with wild-type GATA1 developed CFU-GEMM (colony-forming unit granulocyte erythroid macrophage megakaryocyte), CFU-GM (CUF granulocyte-macrophage) and BFU-E (burst-forming unit erythroid) colonies in Methocult. GATA1 mutation, unlike STAG2 mutation, inhibited the formation of CUF-GEMM and BFU-E colonies. The number of CFU-GM colonies in T21-2GS was significantly reduced compared to T21-2G (Fig. 1C, p=0.002). Lineage expansion and immunophenotyping of these HSPCs in megakaryocyte-specific media showed that these cells expressed markers closely resembling DS-ML immunophenotype. Of note, the myeloid markers, CD13 and CD11b are the only two markers expressed on majority of DS-ML blasts compared to TAM blasts (Karandikar et al., 2001) (Yumura-Yagi et al., 1992). The percentage of CD13 and CD11b expressing cells was higher in megakaryoblasts expanded from iPSC lines with STAG2 GATA1 double mutant (Fig. 1D). The number of cells expressing CD117, a stem cell marker shown recently to be involved in DS-ML progression, were highest in T21-1GS and T21-2GS lines when compared to their respective isogenic family of GATA1 mutant lines. Conclusion GATA1s and STAG2 knockout co-operated to increase the megakaryoid population and the percentage of cells expressing DS-ML markers. We have developed a model system representing DS-ML, which can be used for understanding the individual and synergistic contribution of these gene mutations in disease initiation and progression. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent stem cells (iPSCs) provides an extraordinary tool for hematological disease modeling of rare disorders such as Down syndrome (DS) associated transient myeloproliferative disorder (TMD). TMD is a preleukemic condition observed in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. Hematopoiesis in the bone marrow (BM) is affected by cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs utilize either 2-dimensional (2D) monolayer of mouse stromal cells or animal tissue derived extracellular matrices. Generation of a 3-dimensional (3D) culture environment attempts to facilitate both cell-cell and cell-matrix interactions during iPSC differentiation. This study reports the development of a 3D culture system for hematopoietic differentiation of iPSCs to model TMD. iPSC colonies were encapsulated in 3D polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) (Fig. 1A) and cultured in maintenance medium (mTeSR1™, Stem Cell Technology) without feeder cells. There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig. 1B). The 3D encapsulation did not have an adverse effect on the viability of the iPSC colonies evaluated by in situ staining with viability dye (Fig. 1C). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG whereas colonies in 2D were more flattened (Fig. 1D). The pluripotency of the 3D encapsulated iPSCs was confirmed alkaline phosphatase staining (purple colonies) and by the presence of >96% population expressing pluripotency markers, Tra-1-60 and SSEA-4 (Fig. 1E). To test the efficiency of the 3D model system to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff Hematopoietic Kit. Following differentiation, immunophenotype analysis of single cells by flow cytometry revealed a 1.7-fold higher CD34+CD45+CD38-CD45RA- cell percentage in 3D hydrogels compared to 2D. Further delineation of sub-populations in HSPC compartment from 2D and 3D hydrogel revealed a 1.9-fold and 2.1-fold higher population of early HSPCs and multipotent progenitors (MPPs) in 3D compared to 2D respectively (Fig 1F, *P We used this 3D system to model TMD by utilizing isogenic iPSCs with disomy 21 (D21), trisomy 21 (T21), and trisomy 21 bearing pathologic mutation in GATA1 (T21-G1). The megakaryoid population in the HSPCs generated by hematopoietic differentiation of 3D encapsulated iPSCs was characterized by the percentage of CD34+CD41+ population within the total CD41+ population, myeloid population as CD18+CD45+ and erythroid population as CD71+CD235+. T21 HSPCs showed increased erythroid and megakaryoid populations as compared to isogenic D21, consistent with the role of trisomy 21 in perturbing hematopoiesis. T21-G1 had elevated megakaryoid (93±6% vs 71±1%,) and myeloid (32±16% vs 8±4%) populations with reduced erythroid (27±12% vs 79±6%) population as compared to T21 HSPCs implicating GATA1s in altered hematopoiesis (Fig. 1H). T21-G1 HSPCs only produced CFU-GM colonies as compared to a high number of CFU-GEMM and BFU-E in T21 and D21 HSPCs (Fig. 1I). The expression of GATA1s in T21-G1 megakaryoid population was confirmed (Fig. 1J). The immunophenotype marker analysis of T21-G1 megakaryoid blasts showed expression of megakaryoid/erythroid antigens (CD41, CD61, CD42b, CD71) along with myeloid markers (CD11b, CD33, CD13) and increased expression of CD56 and CD117 consistent with TMD patients (Fig. 1K). In conclusion, our cost-effective tunable 3D hydrogel system promoted hematopoietic differentiation of iPSCs and generated TMD model mimicking the salient features of the disease. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

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 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 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.

Pediatric acute myeloid leukemia (AML) patients possessing rearrangement of the KMT2A (previously known as MLL) gene on 11q23 constitute a subclass with a particularly poor prognosis. The five-year survival rate for these patients is only about 44% due to poor response to conventional chemotherapy and frequent early relapse. Aberrant epigenetic modifications play an important role in leukemogenesis in KMT2A-rearranged leukemia. Accordingly, several epigenome modifying drugs have been tested in preclinical studies of KMT2A-rearranged leukemia. Acknowledging the co-regulatory effects of DNA methylation and histone modifications in determining chromatin structure and governing gene expression, we combined DNA hypomethylating agent azacitidine with histone deacetylase inhibitor panobinostat in the hopes of achieving greater efficacy. We showed that this epigenetic drug combination was more efficacious than single agents using cell line derived xenograft models of pediatric AML (Gopalakrishnapillai et al., Leuk Res, 2017). We evaluated the efficacy of this epigenetic drug combination in patient-derived xenograft models of KMT2A rearranged pediatric AML and observed that similar to MV4;11 model, this combination induced complete remission in NTPL-146 model with KMT2A-MLLT1 fusion (Fig. 1A, P Methylation patterns between treatment groups were discriminated using an ordinate analysis technique of non-metric multidimensional scaling (NMDS) (Fig. 1B). CpG methylation profiles were compared among the four groups analyzed to isolate patterns conserved within groups while also differing between groups. The first two component axes were plotted to locate the individual sample points in a 2D plane. Samples from distinct PDX models undergoing similar treatment clustered together. The panobinostat-treated samples showed minimal differences compared to the control, while the azacitidine-treated samples clustered away. Interestingly, the samples treated with the combination, did not overlap with either treatment, indicating that although panobinostat alone showed minimal impact on methylation patterns, panobinostat together with azacitidine produced a distinct methylation pattern. Venn intersection sets of statistically significant differentially methylated CpG sites in the 3-way analyses derived from the control group comparisons showed 2086 CpG sites exclusively altered in the combination treatment (Fig. 1C). In order to determine the effect of the combination treatment on global methylation, the differences in methylation load (dML) per each CpG site between control and the combination treatment were summed across 1MB genome intervals and the distribution of these dML was plotted (Fig. 1D). There was a strong shift in methylation signal, with the majority of the intervals being hypomethylated in the treatment group compared to the control. Although global hypomethylation was observed in combination treatment, the most statistically significant CpG sites were hypermethylated in the combination treatment compared to the control as seen in the volcano plot in which log fold-change was plotted against the p-value (Fig. 1E). Circular ideogram presented with a mean subtraction of CpG methylation scores to calculate a summation methylation load score across chromosomal domains (Fig. 1F). The correlative association between top CpG sites is shown as arcs tracking the highest correlation for each CpG site. Gene labels indicate the positions of the top 60 CpG sites, with green and red indicating higher methylation in control and in combination treatment respectively. In conclusion, we have identified differential methylation patterns following in vivo treatment of KMT2A rearranged pediatric AML xenograft models with azacitidine and panobinostat combination compared to azacitidine alone. These methylation changes are likely to influence the increased survival seen in mice receiving combination treatment. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Marsh: Genome Profiling LLC: Current Employment. Barwe: Prelude Therapeutics: Research Funding.

Background: Down syndrome is known for its leukemia predisposition effects. Children with Down syndrome myeloid leukemia who fail chemotherapy, have a poor outcome. No targeted therapies are available for this population. We have generated and extensively characterized several patient-derived xenograft (PDX) models of Down syndrome AML (Barwe et al., 134:2683, Blood, 2019). We evaluated the efficacy of a combination of DNA hypomethylating agent, azacitidine and Bcl2 inhibitor, venetoclax in PDX model of Down syndrome AML. Although this drug combination is used in the clinic for the treatment of adult AML, their combinatorial mechanism of action is not well known. Methods: NTPL-386 (3 x 10 6 cells) were injected intravenously in NSG-SGM3 mice. Disease progression was monitored by determination of the percentage of human chimerism in peripheral blood by flow cytometry. Mice were randomly assigned to treatment groups (n=5) on day 13 when human CD45+ cells were detectable in blood. Azacitidine (2.5 mg/Kg) was administered intraperitoneally on days 13-19 and venetoclax (100 mg/Kg) was delivered orally on days 13-40 (Fig. 1A). Mice were monitored daily for pre-determined experimental endpoints and were euthanized when any of the endpoints were attained. Kaplan-Meier survival plots were generated. AML cells were harvested from treated mice and lysed, RNA was isolated and sequenced. Pathway analysis was performed to identify the mechanism of action of these two drugs in combination. Results: Azacitidine treatment for one week increased median survival by 8 days compared to untreated mice (Fig. 1B). Venetoclax treatment for 4 weeks was more effective than azacitidine and extended survival by 27 days (P To understand the mechanism of synergism between these two drugs, we conducted transcriptome analysis on cells harvested from mice (n=3 per group) receiving venetoclax and azacitidine, alone or in combination. The log fold changes for each treatment with respect to untreated were calculated and filtered based on FDR>0.05. The genes that were differentially regulated in the combination treatment (Fig. 1C, blue circle in the Venn diagram) were subjected to ToppGene analysis. A heatmap of the differentially regulated genes shows clustering of the replicates from individual mice (Fig. 1D). 'Hematopoietic cell lineage' and 'cytokine signaling in immune system' were the top modulated pathways in the combination treatment. A heatmap of the logFC of the differentially regulated genes from this pathway showed the modulation of these genes in the combination in comparison with individual drug treatment (Fig. 1E). The differentially regulated genes in the combination treatment that showed high variance from the range defined by individual drug treatment belonged to one of three categories - cell surface proteins, cytokines, and stem cell markers. These data indicate that the synergistic downregulation of cytokine signaling is likely responsible for the combinatorial effect of venetoclax and azacitidine. Conclusion: The combination of venetoclax and azacitidine was effective in prolonging survival in a highly refractory Down syndrome AML PDX model (NTPL-386) generated in the laboratory. The top genes differentially regulated by the combination treatment which varied the most in magnitude from the individual drug treatment identified genes that are likely to contribute to the synergism between the two drugs. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Hematopoietic stem and progenitor cells located in the bone marrow lay the foundation for multiple lineages of mature hematologic cells. Bone marrow niches are architecturally complex with specific cellular, physiochemical, and biomechanical factors. Increasing evidence suggests that the bone marrow microenvironment contributes to the pathogenesis of hematological neoplasms. Numerous studies have deciphered the role of genetic mutations and chromosomal translocations in the development hematologic malignancies. Significant progress has also been made in understanding how the cellular components and cytokine interactions within the bone marrow microenvironment promote the evolution of hematologic cancers. Although the extracellular matrix is known to be a key player in the pathogenesis of various diseases, it's role in the progression of hematologic malignancies is less understood. In this review, we discuss the interactions between the extracellular matrix and malignant cells, and provide an overview of the role of extracellular matrix remodeling in sustaining hematologic malignancies.

The mesothelin (MSLN) protein is overexpressed in many solid tumors and is considered a viable immunotherapeutic target. We recently demonstrated that MSLN is aberrantly expressed on the blast surface in approximately 1/3 of AML cases and lacks expression in normal hematopoiesis, making it an attractive target in AML. While the exact function of MLSN is unknown, multiple studies, including its recently described role in the development of surgical adhesions, implicate it in cell adhesion in both healthy and malignant cells. We hypothesized that MLSN expression in AML may be involved in extramedulllary disease (EMD). We correlated MLSN transcription expression with EMD status and utilized xenograft models to evaluate MSLN's role in AML in vivo. A total of 1,038 patients with de novo AML (age 0-29 years) enrolled on COG AAML1031 with available transcriptome, clinical, and biologic data were included for analysis. Transcriptome sequencing was performed on diagnostic bone marrow or peripheral blood specimens and gene expression was quantified as transcripts per million (TPM). The cohort with MLSN positive (MSLN+) AML (n=359) was defined as patients with MSLN overexpression, 5 TPM, and Our analysis demonstrated a striking association between MSLN expression and EMD. Among the MSLN+ cohort, the rate of EMD was significantly higher compared to the MSLN- cohort (27.8% vs. 18.8%, p=0.001; Table 1). When stratified by EMD type, we observed a similar pattern with higher rates of both non-CNS and CNS EMD in MSLN+ AML. Non-CNS EMD occurred in 17% of MSLN+ vs. 12.4% of MSLN- AML (p=0.042). The rate of CNS positive (CNS3) disease was significantly higher in the MSLN+ cohort compared to the MSLN- cohort (13.2% vs. 7.6%, p=0.004; Table 1). While MSLN+ cases made up 35% of the cohort, they accounted for 47% of the CNS positive cases. Of interest, MSLN+ disease was also associated with significantly higher occurrence of CNS2 disease compared to MSLN- (26.4% vs. 19.1% respectively, p=0.008; Table 1). In patients with very high MSLN expression (100 TPM, n=112), 29% (n=32) of patients had EMD (n=23 non-CNS, n=9 CNS). Notably, among the cohort in this very high expressing group that lacked EMD at diagnosis but who experienced relapse (n=35), 38% (n=13) had EMD at relapse. Among patients with relapsed/refractory (R/R) AML (n=486), MSLN overexpression was also highly associated with EMD. Although MSLN+ cases comprised 35% of R/R cases, they accounted for 53% of EMD cases. The rate of EMD was 40.8% in MSLN+ cases vs. 18.9% of MSLN- cases (p We evaluated the role of MLSN in EMD and its potential function in engraftment in vivo with xenograft models. A higher incidence of EMD was detected in MSLN+ xenografts; 90% (9/10) of Nomo-1 xenografts developed abdominal myeloid sarcomas compared to 30% of Nomo-1KO-MSLN (p Our findings demonstrate that MSLN is highly implicated in EMD and our xenograft studies further support the hypothesis that MSLN may be involved in cell adhesion and establishing sites of disease. EMD can be very challenging to treat, and in the setting of recurrence often indicates incurable disease, thus novel therapeutic strategies for this group of patients are urgently needed. Our findings support additional studies of how MSLN may be functionally implicated in the development of EMD as well as clinical trials that evaluate MSLN targeting in AML. Disclosures No relevant conflicts of interest to declare.

Mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT) are important processes in kidney development. Failure to undergo MET during development leads to the initiation of Wilms tumor, whereas EMT contributes to the development of renal cell carcinomas (RCC). The role of calcium regulators in governing these processes is becoming evident. We demonstrated earlier that Na+/Ca2+ exchanger 1 (NCX1), a major calcium exporter in renal epithelial cells, regulates epithelial cell motility. Here, we show for the first time that NCX1 mRNA and protein expression was down-regulated in Wilms tumor and RCC. Knockdown of NCX1 in Madin-Darby canine kidney cells induced fibroblastic morphology, increased intercellular junctional distance, and induced paracellular permeability, loss of apico-basal polarity in 3D cultures, and anchorage-independent growth, accompanied by expression of mesenchymal markers. We also provide evidence that NCX1 interacts with and anchors E-cadherin to the cell surface independent of NCX1 ion transport activity. Consistent with destabilization of E-cadherin, NCX1 knockdown cells showed an increase in β-catenin nuclear localization, enhanced transcriptional activity, and up-regulation of downstream targets of the β-catenin signaling pathway. Taken together, knockdown of NCX1 in Madin-Darby canine kidney cells alters epithelial morphology and characteristics by destabilization of E-cadherin and induction of β-catenin signaling.

Aberrations in epigenetic modifications contribute to leukemogenesis in childhood acute myeloid leukemia (AML). We combined DNA hypomethylating agent azacitidine with histone deacetylase inhibitor panobinostat in preclinical models of childhood AML. Synergistic cytotoxic effect upon treatment with azacitidine and panobinostat with combination indices < 1.0 was observed. Azacitidine and panobinostat increased median survival by 26 and 6 days respectively in MV4;11 xenografted mice. Mice treated with both drugs showed a drastic reduction in leukemic burden leading to complete remission sustained for the duration of the experimental period lasting more than 519 days. Reduced leukemic burden and prolonged survival was also observed in AML-193 xenografted mice treated with azacitidine-panobinostat combination. Differential gene expression profiling was performed on AML cells treated with azacitidine, panobinostat or azacitidine-panobinostat combination. Functional mapping of transcripts uniquely regulated by the azacitidine-panobinostat combination in MV4;11 cells identified p53 as an upstream regulator. A comparison of the uniquely modulated transcripts by azacitidine-panobinostat combination in MV4;11 cells versus AML-193 and THP-1 cells, bearing mutated p53, also revealed p53 as the topmost upstream regulator. Finally, expression of mutant p53 in MV4;11 cells reduced sensitivity to azacitidine-panobinostat combination, suggesting that p53 may be a predictor of response to epigenetic therapy in pediatric AML.

Epidermal growth factor (EGF) signaling regulates cell growth, proliferation, and differentiation. Upon receptor binding, EGF triggers cascades of downstream signaling, including the MAPK and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways. Aberrant expression/activation of EGFR is found in multiple human cancers, including medulloblastoma, the most prevalent pediatric brain cancer, and often has been associated with metastasis, poor prognosis, and resistance to chemotherapy. Na,K-ATPase is an ion pump well known for its role in intracellular ion homeostasis. Recent studies showed that Na,K-ATPase also functions as a signaling platform and revealed a role in EGFR, MAPK, and PI3K signaling. While both EGFR and Na,K-ATPase seem to modulate similar signaling pathways, cardiac glycosides that are steroid-like inhibitors of Na,K-ATPase, exhibit antiproliferative and proapoptotic properties in cancer cells. Thus, we sought to better understand the relationship between EGF and cardiac glycoside signaling. Here, we show that in medulloblastoma cells, both EGF and ouabain activate Erk1/2 and PI3K/Akt signaling. Nevertheless, in medulloblastoma cells ouabain did not transactivate EGFR as has been reported in various other cell lines. Indeed, ouabain inhibited EGF-induced Erk1/2 and Akt activation and, moreover, prevented EGF-induced formation of actin stress fibers and cell motility, probably by activating a stress signaling response. Na,K-ATPase has been proposed to act as a signaling scaffold and our studies suggest that in medulloblastoma cells Na,K-ATPase might act as a check point to integrate EGF-associated signaling pathways. Thus, Na,K-ATPase might serve as a valid target to develop novel therapeutic approaches in tumors with aberrant activation of the EGFR signaling cascades.

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent cells (iPSCs) holds a great potential in development of cell therapies, modeling hematological malignancies, and testing new drugs. Hematopoiesis is regulated by bone marrow (BM) microenvironmental factors such as BM architecture, and cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs rely on cell-cell interaction (embryoid body formation and feeder cell co-cultures) or cell-matrix interaction (ECM coated dishes). Generation of a 3-dimensional (3D) culture environment attempts to capture both cell-cell and cell matrix interactions in studying the differentiation of the iPSCs. This study reports development of a 3D culture system for hematopoietic differentiation of iPSCs. iPSC colonies were encapsulated in 3D Matrigel or polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) and cultured in feeder-free cell culture and maintenance medium (mTeSR1™, Stem Cell Technology). There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig 1A). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG compared to Matrigel, whereas colonies in 2D were more diffused. Viability of the encapsulated colonies was evaluated in situ using cytotoxicity kit. The 3D encapsulation did not have an adverse effect on the viability and growth of iPSCs (Fig 1B). The encapsulated iPSCs maintained the pluripotent phenotype in 3D as assessed by TRA-1-60 staining (Fig 1C). To test the efficiency of these 3D model systems to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff™ Hematopoietic Kit (Fig 1D). Following differentiation, the single cells were collected and immunophenotype analysis was performed by flow cytometry. Above 50% population stained positive for HSPC surface marker CD34 both in PEG hydrogel and Matrigel (Fig 1E). To test the robustness of the 3D system in inducing hematopoietic differentiation, we encapsulated the isogenic trisomic 21 (Tri21) iPSCs and subjected to hematopoietic differentiation and flow cytometry analysis as described above. As reported previously in 2D system (Banno et al., 2016, Cell Reports), Tri21 iPSCs showed higher percentage of hematopoietic commitment into erythroid and megakaryocytic lineages compared to disomic 21 (Di21), as characterized by the presence of respective surface markers (Fig 1F). In conclusion, 3D culture system was suitable for inducing hematopoietic differentiation of iPSCs in isogenic disomic and trisomic lines. The 3D model can be used to generate patient-specific HSPCs. Figure 1 Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) remains the type of pediatric leukemia with poorest outcome. Despite maximally intensive therapy, approximately 20% of patients experience recurrent disease. Novel targeted therapies are needed to improve survival. We recently identified that mesothelin, a well-validated target in some cancers, is also highly expressed in a subset of pediatric AML samples (Tarlock et al., Blood, 128:2873, 2016). Considering that it is not expressed in normal tissues in children (Fan et al., Blood, 130:3792, 2017), MSLN is a viable target for immunotherapies such as Bispecific T-cell Engaging antibodies (BiTEs) that combine antibody single chain variable (scFv) regions targeting a cancer antigen and the T-cell co-receptor CD3. We designed and tested the efficacy and specificity of BiTEs targeting MSLN in disseminated xenograft models of pediatric AML. Using scFv sequences derived from Amatuximab, which recognizes the N-terminal domain of the GPI-linked ectodomain of MSLN, targeting region 1 of MSLN, and from Blinatumomab/AMG-330 targeting CD3, we engineered and expressed two kinds of BiTE molecules - a canonical BiTE and an IgG BiTE, a larger molecule with improved serum half life in vivo. To evaluate the specificity and efficacy of canonical BiTEs, MV4;11-MSLN cell line was generated by lentiviral transduction of parental MV4;11 cells which do not constitutively express MSLN (Fig. 1A, B). These two cell lines were injected i.v. into NSG-SGM3 mice. Once engraftment was confirmed, purified human T cells (3 x 106) were injected to act as effector cells. Mice were then treated with the canonical αMSLN-αCD3 BiTE at a dose of 3 mg/kg/day daily for 6 days. A cohort of mice that were untreated or received BiTE or T-cell infusion only served as controls. Mice from both treated and untreated groups had to be euthanized when they presented with distended abdomens due to myeloid sarcomas and no significant differences in survival were observed. Post euthanasia, bone marrows were flushed and evaluated for the percentage of AML cells (human CD45+CD33+) and T cells (human CD45+CD3+). We observed that the αMSLN-αCD3 BiTE was effective in promoting T-cell activation (based on high T-cell counts compared to mice injected with T-cells alone) and greatly reducing leukemic burden in mice injected with MV4;11 cells engineered to express MSLN (Fig. 1C, D). Similar results were obtained using BiTEs targeting a different MSLN epitope. No T-cell expansion and anti-leukemic effect was observed in mice engrafted with parental MV4;11 cells. Although, there were no significant differences between the median survival of untreated and treated miceThese data highlight the specificity and efficacy of the aMSLN-CD3 BiTEs. Among a panel of 8 AML patient-derived xenograft (PDX) lines generated in the laboratory, NTPL-146 bearing MLL-ENL fusion was found to have endogenous MSLN expression (Fig. 1E). We evaluated the efficacy of αMSLN-αCD3 canonical BiTE (3 mg/Kg Qdx6) against NTPL-146 PDX line in NSG-B2m mice by transfusing human CD3+ T-cells to act as effector cells. A Kaplan-Meier survival plot based on the time when each mouse reached experimental end-point (reduced body weight greater than 20%, impaired mobility, hind limb paralysis) showed that the survival benefit for mice receiving BiTE in the presence of human T-cells (4/6 mice survived at the end of experiment) greatly exceeded the efficacy of T-cells alone (22-day improvement in median survival with no surviving mice), or BiTE treatment alone (no improvement in survival) compared to untreated mice (Fig. 1F, P The efficacy of canonical vs IgG BiTEs was evaluated in MV4;11-MSLN xenografted mice. Mice were dosed Qd5x3 for canonical BiTE and Q7dx3 for IgG BiTE as shown (Fig. 1G). IgG BiTE treatment along with T-cell infusion significantly prolonged survival in mice transplanted with MV4;11-MSLN (Fig. 1H), suggesting that IgG BiTE was far more efficacious than canonical BiTEs (P Figure 1 Disclosures Kaeding: Celgene: Employment.

Children with Down syndrome (DS) are at a 500-fold increased risk for developing acute myeloid leukemia (AML) before they reach five years of age. DS-AML blasts have hypersensitivity to chemotherapeutic drugs such as cytarabine and daunorubicin. However, therapy-induced toxicity results in greater morbidity and remains a major barrier in attaining higher survival rate. Thus, alternate therapy approaches to minimize toxicity and increase efficacy are needed. Patient-derived xenograft (PDX) models of DS-AML are very limited and have not been used for preclinical studies. We generated three distinct disseminated PDX models by successful engraftment and serial passage of primary DS-AML blasts in NSG-B2m mice. DS-AML is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated mutant GATA1s protein. Accordingly, we identified GATA1 mutations and GATA1s protein (Fig. 1) in the DS-AML PDX lines. In addition to GATA1 mutation, whole exome sequencing also identified mutations in one or more co-operating mutations reported previously in DS-AML (Table 1). These PDX lines were extensively characterized and authenticated to be highly concordant with the primary patient sample with respect to immunophenotype (Table 2) and transcriptome analysis (by targeted sequencing using Archer FusionPlex HemeV2 panel), signifying the validity of the PDX models. We evaluated the efficacy of cytotoxic chemotherapy and epigenetic therapy in mice transplanted with DS-AML PDX lines. Cytotoxic chemotherapy comprised daunorubicin 1.5 mg/Kg Qxd3 i.v. and cytarabine 50 mg/Kg Qxd5 i.p. Epigenetic therapy consisted of DNA hypomethylating agent azacitidine and HDAC inhibitor panobinostat, alone or in combination. NSG-B2m mice were injected with DS-AML PDX lines intravenously. Disease progression and engraftment of human cells was monitored by periodic determination of the percentage of human leukemic cells versus mouse cells in the peripheral blood by staining with species-specific fluorescent antibodies followed by flow cytometry. The mice were randomized into 5 treatment groups - 1) cytotoxic chemotherapy, 2) vehicle (5% dextrose), 3) azacitidine (2.5 mg/Kg), 4) panobinostat (2.5 mg/Kg), and 5) combination of azacitidine + panobinostat (2.5 mg/Kg each). The epigenetic drugs were dosed with four cycles lasting five days a week with two days rest. One-week rest period was included between cycles 2 and 3. Chemotherapy prolonged survival by 11 and 23 days respectively in mice injected with NTPL-386 and NTPL-662. Single agent azacitidine and panobinostat prolonged survival by 48 and 31 days respectively in NTPL-60 mice. Mice transplanted with NTPL-386 showed enhanced survival by 29 and 21 days when treated with azacitidine and panobinostat alone. NTPL-662 study is ongoing and so far panobinostat treated mice showed 10 day increase in cell survival, while both azacitidine and azacitidine + panobinostat groups are alive and healthy. Azacitidine was more efficient than panobinostat in inducing remission and increasing the median survival compared to vehicle treated mice in all 3 models (Fig. 2A-C). Mice treated with the combination of azacitidine and panobinostat showed the greatest benefit and survived 127 days (for NTPL-60) and 55 days (for NTPL-386). Although azacitidine and panobinostat synergized to reduce leukemic burden and prolong survival in both PDX lines, there was a remarkable difference between the responses. Epigenetic therapy appeared to be more effective than chemotherapy. In conclusion, using DS-AML PDX models generated and characterized in-house, we show the efficacy of epigenetic therapy in comparison to chemotherapy. Disclosures Druley: Washington University: Employment; ArcherDX Inc.: Employment, Equity Ownership.

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 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.

Down syndrome (DS) is recognized as one of the most important leukemia-predisposing syndromes. Specifically, 1-2% of DS children develop acute myeloid leukemia (AML) prior to age 5. AML in DS children (DS-AML) is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated mutant GATA1 (GATA1s). Trisomy 21 and GATA1s together induce a transient myeloproliferative disorder (TMD) exhibiting pre-leukemic characteristics. Approximately thirty percent of these cases progress into DS-AML by acquisition of additional somatic mutations in a step-wise manner. We employed disease modeling in vitro by the use of customizable induced pluripotent stem cells (iPSCs) (7, 8) to generate a TMD model. Isogenic iPSC lines derived from the fibroblasts of a DS patient with trisomy 21 and with disomy 21 were used. We also obtained DS2-iPS10 (iPSCs derived from DS patient fibroblast) from Prof. George Daley, Children's Hospital, Harvard University (Boston, MA). CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system with the indicated guide sequence (Fig. 1A) was used to introduce clinically relevant GATA1 mutation in both disomic and trisomic iPSC lines. A representative plot of TIDE (Tracking of Indels by Decomposition) analysis showing 98% allelic mutation frequency of a clone with 2 bp deletion at chromosomal level (Fig. 1B) correlated with sequence analysis using Basic Local Sequence Alignment Tool (BLAST) and Sanger sequencing chromatogram (Fig. 1C). This mutation resulted in the disruption of first initiation codon and thus prevented the production of full length GATA1 protein, while allowing the usage of second initiation codon at 84th position to generate GATA1s. GATA1 and GATA1s are not expressed in iPSCs. To determine the expression of GATA1s, we differentiated these mutant iPSC lines into hematopoietic stem cell progenitors (HSPCs) using hematopoietic differentiation kit (StemCell Technologies) following a protocol depicted in Fig. 1D. The HSPCs derived from two distinct clones of trisomic iPSCs showed expression of full-length GATA1 protein and GATA1 mutant HSPCs lacked the expression of full-length GATA1 as expected (Fig. 1E). These HSPCs expressed GATA1s. Given that trisomy 21 promotes hematopoietic differentiation, an increase in the percentage of erythroid, megakaryoid and myeloid population was observed in trisomy 21 HSPCs with full length GATA1 (Fig. 1F, compare bars 1 and 3 in each category). The expression of GATA1s reduced erythroid lineage cells whereas it augmented megakaryoid and myeloid lineages in both disomy 21 (compare red and blue bars 1 and 2) and trisomy 21 backgrounds (compare bars 3 and 4). HSPCs derived from trisomy 21 iPSCs with GATA1s exhibited more megakaryoid expansion compared to the GATA1s in disomy 21 background (Fig. 1F, compare bars 2 and 4), in agreement with the synergistic function of trisomy 21 and GATA1s in promoting TMD. Transplantation of HSPCs derived from GATA1 mutated trisomic iPSCS into NSG-SGM3 mice showed the presence of human CD45+ cells in peripheral blood at 12 weeks post cell injection (Fig. 1G). In conclusion, we have developed a model system representing TMD, which can be used for step-wise modeling of Down-syndrome AML by introducing additional mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Background We previously discovered that the epigenetic drug combination - azacitidine (aza, DNMTi) and panobinostat (pano, HDACi) can sensitize ALL cells to chemotherapy (Quagliano et al., Leuk Res, 44:101, 2017). This sensitization occurred through a decrease in cellular adhesion and a modulation of the cell surface expression of the tetraspanin protein CD81 (Quagliano et al., Blood, 132:3957, 2018). This study aims to elucidate the molecular mechanism by which CD81 surface modulation by pre-treatment with aza/pano leads to chemosensitization. Methods CD81 knockout Nalm6 (CD81KO) cells were generated by CRISPR/Cas9 mutagenesis. IHC was performed using anti-human mitochondria antibody on femurs harvested from NSG-SGM3 mice at 72 h post transplantation with WT or CD81KO cells. For adhesion assay, ALL cells stained with VPD450 were co-cultured with Saos-2 cells for 24 h. Unbound cells were washed, and bound cells were collected for flow cytometry analysis. CD19 surface expression was determined after 48 h treatment with aza/pano (500 nM/1.5 nM) using flow cytometry. For western blot analysis, aza/pano treated cells were transferred on to Saos-2 monolayers and treated with Ara-C (30 nM) for 16 h. Results CD81KO cells not only had decreased homing and engraftment visualized by reduced ALL cell presence in bone marrow (Fig. 1A), but also had 45% reduction in cellular adhesion to osteoblasts compared to WT Nalm6 cells (Fig. 1B), indicating that CD81 downregulation results in reduced bone marrow interactions. To investigate the signaling effects downstream of aza/pano-mediated CD81 modulation, we first tested if CD19 was affected due to its prominent role in the formation of the B-cell co-receptor and because CD19 requires CD81 for proper membrane trafficking (Cherukuri et al., J Immunol, 172:370, 2004). CD19 surface expression was reduced in aza/pano-treated cells and completely knocked out in CD81KO cells (Fig. 1C). CD19 is known to prolong and amplify the activation of Bruton's tyrosine kinase (BTK), so we also analyzed the phosphorylation of BTK in aza/pano-treated cells and found it to be reduced by 63% (Fig. 1D). This reduction in BTK phosphorylation may be responsible for the decrease in cell adhesion following treatment with aza/pano because prior studies observed reduced adhesion following BTK inhibition (Herman et al., Clin Cancer Res, 21:4642, 2015). Taken together, these data suggest that aza/pano induced reduction in cell adhesion was mediated by downregulation of CD81 and BTK dephosphorylation. We studied how the expression of p53 and its target BCL2 associated X protein (BAX), which are known to be induced by Ara-C, were affected following pre-treatment with aza/pano. Cells with Ara-C treatment alone or the aza/pano pre-treatment both had minor induction of p53 expression compared to control, while aza/pano pre-treated cells that received Ara-C had further increase in p53 protein (1.7-fold increase compared to Ara-C alone, Fig. 1E). Bax mRNA and protein were increased 4.2-fold in aza/pano-treated cells following Ara-C treatment compared to Ara-C alone. Increased Bax expression following Ara-C treatment in aza/pano pre-treated cells was accompanied by a 3.9-fold increase in cleavage of caspase-3 compared to cells treated with Ara-C alone, which in turn causes cleavage of Poly-ADP Ribose Polymerase (PARP) (Fig. 1E). Both p53 and Bax protein levels were higher in CD81KO cells than in WT cells (Fig. 1E), suggesting that aza/pano-mediated reduction of CD81 is involved in inducing the expression of p53. CD81KO cells that were treated with Ara-C also had further increase in p53 and Bax expression compared to Ara-C treated WT cells (Fig. 1E), consistent with an increased sensitivity to chemotherapy. To confirm the role of BTK inhibition in the induction of p53, Nalm6 cells were treated with the BTK inhibitor fenebrutinib for 24 h (10 nM). Following treatment, expression of p53 was induced and Bax expression was 2.6-fold higher in treated cells compared to untreated cells (Fig. 1E). Taken together, these data suggest that modulation of the surface expression of CD81 and the phosphorylation of BTK by aza/pano induces sensitization via upregulation of p53 and overexpression of Bax. Conclusion We identify a novel mechanism by which aza/pano treatment induces chemosensitization by reducing cell adhesion via modulating CD81 surface expression, reducing BTK phosphorylation, and inducing p53. Figure 1 Disclosures No relevant conflicts of interest to declare.

Precursor T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogenous hematologic malignancy resulting from accumulation of molecular lesions in a multistep process. Although survival rates have improved considerably, event free survival for patients with T-ALL are generally inferior compared to B-cell ALL. Genetic alterations are important determinants of responsiveness to therapy and serve as targets for molecularly tailored therapies. More than 75 chimeric fusion genes have been reported in T-ALL, the majority of which encode factors involved in transcriptional regulation, while only a smaller percentage codes for tyrosine kinases. We report a case of relapsed T-ALL, despite low risk stratification at the time of diagnosis, harboring a novel fusion protein SPTAN1-ABL1. Primary bone marrow specimen collected at diagnosis was transplanted in NSG-B2m mice and propagated as a patient-derived xenograft (PDX) line. For transcriptomic characterization, RNA isolated from primary and PDX samples was subjected to error-corrected targeted next-generation sequencing using ArcherDX FusionPlex HemeV2 kit. Bioinformatics analysis identified the novel SPTAN1-ABL1 gene fusion in which exon 2 of SPTAN1 was fused with exon 4 of ABL1. This fusion was confirmed by Sanger sequencing. Translation of the fusion product sequence showed in-frame fusion leading to the generation of a chimeric protein containing N-terminal SPTAN1 and C-terminal ABL1 with intact kinase domain. SPTAN1 encodes non-erythryocytic-1-spectrin-alpha protein, an actin-binding protein, with N-terminal domain possessing oligomerization activity. Because oligomerization of ABL1 promotes its kinase activity, it is possible that SPTAN1-ABL1 possesses constitutive kinase activity. The full-length SPTAN1-ABL1 fusion protein was cloned in a mammalian expression vector and expressed in BaF3 cells. SPTAN1-ABL1 fusion was detected at similar allelic frequencies in primary and PDX samples indicating the concordance between the two. Furthermore, treatment of engrafted mice with dasatinib (Qd10, 5 mg/Kg, p.o.) significantly prolonged survival compared to untreated mice (n=5 each, P Citation Format: Anilkumar Gopalakrisnapillai, Erin Crowgey, Demetria Ruhl, Darcy Hamill, Nitin Mahajan, Todd Druley, E. Anders Kolb, Sonali P. Barwe. Identification of a novel fusion protein SPTAN1-ABL1 in a child with T-cell acute lymphoblastic leukemia: Functional characterization and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-322.

Ionophores are lipid-soluble organic molecules that disrupt cellular transmembrane potential by rendering biologic membranes permeable to specific ions. They include mobile-carriers that complex with metal cations and channel-formers that insert into the membrane to form hydrophilic pores. Although mobile-carriers possess anticancer properties, investigations on channel-formers are limited. Here, we used the channel-forming ionophore gramicidin A to study its effects on the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional treatments. We found that gramicidin A reduced the in vitro viability of several RCC cell lines at submicromolar concentrations (all IC50 < 1.0 μmol/L). Gramicidin A exhibited similar toxicity in RCC cells regardless of histologic subtype or the expression of either the von Hippel-Lindau tumor suppressor gene or its downstream target, hypoxia-inducible factor-1α. Gramicidin A decreased cell viability equal to or greater than the mobile-carrier monensin depending on the cell line. Mechanistic examination revealed that gramicidin A blocks ATP generation by inhibiting oxidative phosphorylation and glycolysis, leading to cellular energy depletion and nonapoptotic cell death. Finally, gramicidin A effectively reduced the growth of RCC tumor xenografts in vivo. These results show a novel application of gramicidin A as a potential therapeutic agent for RCC therapy. Mol Cancer Ther; 12(11); 2296–307. ©2013 AACR.

Generation of orthotopic xenograft mouse models of leukemia is important to understand the mechanisms of leukemogenesis, cancer progression, its cross talk with the bone marrow microenvironment, and for preclinical evaluation of drugs. In these models, following intravenous injection, leukemic cells home to the bone marrow and proliferate there before infiltrating other organs, such as spleen, liver, and the central nervous system. Moreover, such models have been shown to accurately recapitulate the human disease and correlate with patient response to therapy and prognosis. Thus, various immune-deficient mice strains have been used with or without recipient preconditioning to increase engraftment efficiency. Mice homozygous for the severe combined immune deficiency (SCID) mutation and with non-obese diabetic background (NOD/SCID) have been used in the majority of leukemia xenograft studies. Later, NOD/SCID mice deficient for interleukin 2 receptor gamma chain (IL2Rγ) gene called NSG mice became the model of choice for leukemia xenografts. However, engraftment of leukemia cells without irradiation preconditioning still remained a challenge. In this study, we used NSG mice with null alleles for major histocompatibility complex class I beta2-microglobulin (β2m) called NSG-B2m. This is a first report describing the 100% engraftment efficiency of pediatric leukemia cell lines and primary samples in NSG-B2m mice in the absence of host preconditioning by sublethal irradiation. We also show direct comparison of the engraftment efficiency and growth rate of pediatric acute leukemia cells in NSG-B2m and NOD/SCID mice, which showed 80-90% engraftment efficiency. Secondary and tertiary xenografts in NSG-B2m mice generated by injection of cells isolated from the spleens of leukemia-bearing mice also behaved similar to the primary patient sample. We have successfully engrafted 25 acute lymphoblastic leukemia (ALL) and 5 acute myeloid leukemia (AML) patient samples with distinct cytogenetic characteristics in NSG-B2m mice, with the purpose of generating pediatric ALL and AML xenografts for preclinical evaluation of drugs. Thus, our data support the use of NSG-B2m mouse model for leukemia engraftment and in vivo preclinical drug efficacy studies.

Nanotechnology approaches have tremendous potential for enhancing treatment efficacy with lower doses of chemotherapeutics. Nanoparticle-based drug delivery approaches are poorly developed for childhood leukemia. Dexamethasone (Dex) is one of the most common chemotherapeutic drugs used in the treatment of childhood leukemia. In this study, we encapsulated Dex in polymeric nanoparticles and validated their anti-leukemic potential in vitro and in vivo. Nanoparticles (NPs) with an average diameter of 110 nm were assembled from amphiphilic block copolymers poly (ethylene glycol) (PEG) and poly (ε-caprolactone) (PCL) bearing pendant cyclic ketals. The blank nanoparticles were non-toxic to cultured cells in vitro and to mice in vivo. Encapsulation of Dex into the nanoparticles (Dex-NP) did not compromise the bioactivity of the drug. Dex-NPs induced glucocorticoid phosphorylation and showed cytotoxicity similar to the free Dex in leukemic cells. Studies using nanoparticles labeled with fluorescent dyes revealed leukemic cell surface binding and internalization. In vivo biodistribution studies showed NP accumulation in the liver and spleen with subsequent clearance of the particles with time. In a pre-clinical model of leukemia, Dex-NPs significantly improved the quality of life and survival of mice compared to the free drug. To our knowledge, this is the first report showing the efficacy of polymeric nanoparticles to deliver Dex to potentially treat childhood leukemia and reveals that low dose of Dex should be sufficient for inducing cell death and improve survival.

Background We showed earlier that prolonged treatment with a combination of azacitidine (DNMT1 inhibitor) and panobinostat (HDAC inhibitor) induced complete remission in a disseminated xenograft model of KMT2A rearranged AML (Gopalakrishnapillai et al., 2017, Leuk Res). Pretreatment with epigenetic drugs has been shown to induce chemo-sensitivity in some malignancies. However, studies on the use of epigenetic drugs in overcoming chemoresistance mediated by the bone marrow microenvironment are limited. The aim of this study was to determine the merit of incorporating epigenetic therapy with chemotherapy in AML treatment regimen and to identify the mechanism by which epigenetic drugs can chemosensitize AML. Methods AML cell lines and PDX lines were co-cultured with HS5 bone marrow stromal cells. Cell viability following drug treatment was assessed by flow cytometry. For determination of cell adhesion, violet proliferation dye stained AML cells were co-cultured with HS5 cells. Following treatment, non-adherent cells were removed by washes with phosphate buffered saline. The number of adherent AML cells was determined by flow cytometry. NSG-B2m mice were engrafted with MV4;11 cells and bled regularly to evaluate disease progression. Once engraftment was confirmed mice were assigned to treatment groups. Epigenetic therapy constituted azacitidine and panobinostat (2.5 mg/Kg each; Qd5). Chemotherapy constituted cytarabine (50 mg/Kg; Qd5) and daunorubicin (1.5 mg/Kg; Qd3). Mice were euthanized when they reached predetermined experimental endpoints. All animal studies were approved by the Institutional Animal Care and Use Committee. Results AML cells in co-culture with HS5 bone marrow stromal cells were less sensitive to chemotherapeutics cytarabine and daunorubicin compared to AML cells in monoculture. This chemoprotection was not achieved when AML cells were cultured in HS5 conditioned media or in Transwell inserts suspended over HS5 monolayers, suggesting that direct cell-to-cell contact was required. MV4;11 cells exposed to epigenetic drugs or cytarabine alone retained 70% viability. Pre-treatment with the epigenetic drug combination of azacitidine and panobinostat prior to cytarabine exposure greatly reduced cell viability in MV4;11 cells harboring KMT2A fusion (Fig. 1A). Similar chemo-sensitization was observed when four distinct KMT2A rearranged PDX lines were used ex vivo. This sensitization was accompanied by reduced binding of AML cells to HS5 cells following treatment with epigenetic drugs (Fig. 1B). We evaluated the efficacy of the epigenetic therapy and chemotherapy combination in disseminated xenograft models of pediatric AML. NSG-B2m mice transplanted with MV4;11 cells via the tail vein were randomly assigned to four groups - 1) vehicle, 2) epigenetic therapy 3) chemotherapy (cytarabine + daunorubicin), and 4) epigenetic therapy followed by chemotherapy. The mice receiving the epigenetic therapy and chemotherapy combination survived significantly longer than mice treated with any other condition (Fig. 1C). To assess the leukemic cell distribution in peripheral blood, bone marrow and spleen following treatment, a cohort of mice receiving epigenetic therapy alone or chemotherapy alone were euthanized a day after treatment concluded. The percentage of leukemic cells in bone marrow and spleen was lower in mice treated with epigenetic therapy than those treated with chemotherapy, consistent with the survival data. Surprisingly, the peripheral blood counts were significantly higher in mice receiving epigenetic drug combination. These results together with our in vitro data indicate that epigenetic therapy induces mobilization of AML cells to the blood stream. This increased availability of AML cells may promote enhanced sensitivity to chemotherapy. Conclusion Our data suggest that direct cell-to-cell contact plays a major role in mediating chemoprotective effects of the bone marrow microenvironment. These chemoprotective effects can be overcome by pretreatment with epigenetic drug combination azacitidine and panobinostat. Epigenetic drugs interfere with AML cell interactions with the microenvironment and dislodge AML cells from their protective niche. Thus, mobilization of AML cells to peripheral blood is a potential mechanism of chemo-sensitization mediated by epigenetic drugs. Disclosures Kolb: Servier: Membership on an entity's Board of Directors or advisory committees; Roche- Genentech: Membership on an entity's Board of Directors or advisory committees.

Introduction Chromosomal rearrangements generating gene fusions are more common in pediatric malignancies compared to adults, and possess diagnostic, prognostic and therapeutic value. Traditionally chromosomal rearrangements including structural variants (SVs) are identified using karyotyping and fluorescence in situ hybridization (FISH); however, these methods are not sensitive for small rearrangements and single nucleotide variants (SNVs). The need to detect these cryptic SV and SNVs in pediatric cancers demands the development of assays to analyze complex RNA molecules. Methods Primary bone marrow samples obtained from Nemours Biobank were used for RNA isolation. Targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was conducted on 30 primary pediatric leukemia samples and their corresponding mouse passaged xenograft samples. RNA data (fastq) was analyzed via a custom cloud environment leveraging ArcherDx Version 5.1.3 software. The gene fusion data produced by the Archer panel was initially correlated with diagnostic FISH data available for each primary sample. Results Ten out of 30 primary bone marrow samples possessed gene fusions detected by routinely tested FISH probes for diagnostic purposes, and including ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and KMT2A. In addition, this approach detected cryptic gene fusions in 10 samples that were negative for chromosomal rearrangements via FISH, including SPTAN1-ABL1, RUNX1-MKL1, NUP98-NSD1, P2RY8-CRLF2 and TCF3-HLF. The remaining 10 samples, which did not possess detectable gene fusions, showed abnormal exon usage and domain duplications for several, key oncogenes along with novel mutations. A comparison of the primary sample and mouse passaged xenograft sample revealed that majority of gene fusions representing the abundant clone remained consistent between the primary and xenograft sample in secondary and tertiary passages. Certain gene fusions representing minor clones appeared and disappeared in xenograft samples and subsequent passages in mice in comparison to the primary patient sample, highlighting the heterogeneity of the disease. Thus, the presence of major driver mutations at similar allelic frequencies in xenografts compared to primary samples and over multiple passages confirms the utility of xenograft models for preclinical drug testing. Discussion Using a novel approach that utilizes targeted error-corrected sequencing, all the aberrations detected by clinical diagnostic testing were verified, plus several novel fusion events were identified with high confidence. This method also validated the concordance between primary and xenograft samples. Characterization of these novel cryptic fusions and exonal variants in leukemogenesis will enable identification of new drug targets and prognostic factors for pediatric leukemia. Citation Format: Sonali P. Barwe, Anilkumar Gopalakrishnapillai, Nitin Mahajan, Todd Druley, E. Anders Kolb, Erin L. Crowgey. Identification of novel fusion genes and expression variants in primary and patient-derived xenograft samples of pediatric leukemia using error-corrected RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2076.

Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.

Na,K-ATPase is a hetero-oligomer of alpha and beta-subunits. The Na,K-ATPase beta-subunit (Na,K-beta) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-beta transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-beta with Na,K-alpha, and the glycine zipper face is involved in the homo-oligomerization of Na,K-beta. Point mutations in the heptad repeat motif reduced Na,K-beta binding to Na,K-alpha, and Na,K-ATPase activity. Na,K-beta TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-beta links ion transport and cell-cell adhesion.