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Preferentially Expressed Antigen in Melanoma (PRAME), a cancer-testis antigen, provides an ideal target for immunotherapy in acute myeloid leukemia (AML). We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a chimeric antigen receptor (CAR) construct encoding a targeting domain based on T-cell receptor (TCR) mimic antibodies that target the peptide-HLA complex. We used the antibody sequence from a previously designed TCR mimic (mTCR) antibody, Pr20, that recognizes the PRAME ALY peptide in complex with HLA-A∗02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary samples from patients with AML and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to appropriate controls, PRAME mTCRCAR T cells demonstrate target-specific and HLA-mediated in vitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. In vivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared with unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with interferon gamma, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.

Purpose: We previously identified mesothelin (MSLN) as highly expressed in a significant fraction of acute myeloid leukemia (AML) but entirely silent in normal hematopoiesis, providing a promising antigen for immunotherapeutic targeting that avoids hematopoietic toxicity. Given that T cells genetically modified to express chimeric antigen receptors (CAR) are effective at eradicating relapsed/refractory acute lymphocytic leukemia, we developed MSLN-directed CAR T cells for preclinical evaluation in AML. Experimental Design: The variable light (VL) and heavy (VH) sequences from the MSLN-targeting SS1P immunotoxin were used to construct the single-chain variable fragment of the standard CAR containing 41-BB costimulatory and CD3Zeta stimulatory domains. The preclinical efficacy of MSLN CAR T cells was evaluated against AML cell lines and patient samples expressing various levels of MSLN in vitro and in vivo. Results: We demonstrate that MSLN is expressed on the cell surface of AML blasts and leukemic stem cell–enriched CD34+CD38− subset, but not on normal hematopoietic stem and progenitor cells (HSPC). We further establish that MSLN CAR T cells are highly effective in eliminating MSLN-positive AML cells in cell line– and patient-derived xenograft models. Importantly, MSLN CAR T cells can target and eradicate CD34+CD38− cells without impacting the viability of normal HSPCs. Finally, we show that CAR T-cell functionality can be improved by inhibition of the ADAM17 metalloprotease that promotes shedding of MSLN. Conclusions: These findings demonstrate that MSLN is a viable target for CAR T-cell therapy in AML and that inhibiting MSLN shedding is a promising approach to improve CAR T-cell efficacy.

As the molecular mechanisms of biological aging become better understood, there is growing interest in identifying interventions that target those mechanisms to promote extended health and longevity. The budding yeast Saccharomyces cerevisiae has served as a premier model organism for identifying genetic and molecular factors that modulate cellular aging and is a powerful system in which to evaluate candidate longevity interventions. Here we screened a collection of natural products and natural product mixtures for effects on the growth rate, mTOR-mediated growth inhibition, and replicative lifespan. No mTOR inhibitory activity was detected, but several of the treatments affected growth rate and lifespan. The strongest lifespan shortening effects were observed for green tea extract and berberine. The most robust lifespan extension was detected from an extract of Pterocarpus marsupium and another mixture containing Pterocarpus marsupium extract. These findings illustrate the utility of the yeast system for longevity intervention discovery and identify Pterocarpus marsupium extract as a potentially fruitful longevity intervention for testing in higher eukaryotes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00418-x.

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.

Fusion oncoproteins are the initiating event in AML pathogenesis, although they are thought to require additional cooperating mutations for leukemic transformation. CBFA2T3-GLIS2 (C/G) fusion occurs exclusively in infants and is associated with highly aggressive disease1-4. Here we report that lentiviral transduction of C/G fusion is sufficient to induce malignant transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) that fully recapitulates C/G AML. Engineered CB HSPCs co-cultured with endothelial cells undergo complete malignant transformation with identical molecular, morphologic, phenotypic and disease characteristics observed in primary C/G AML. Interrogating the transcriptome of engineered cells identified a library of C/G fusion-specific targets that are candidates for chimeric antigen receptor (CAR) T cell therapy. We developed CAR-T cells directed against one of the targets, FOLR1, and demonstrated the pre-clinical efficacy against C/G AML while sparing normal hematopoiesis. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Moreover, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.

Effective immunotherapy for acute myeloid leukemia (AML) has been limited by the lack of AML-restricted targets (expression in AML but silent in normal hematopoiesis). Current immunotherapies targeting lineage markers CD33 and CD123 (if effective) would lead to prolonged myelosuppression or myeloablation, requiring stem cell transplantation to restore hematopoiesis after treatment. In search for AML-restricted targets, we interrogated the AML transcriptome of nearly 3000 cases in pediatric and young adults and contrasting it to normal bone marrow and peripheral blood CD34+ samples. This extensive discovery effort has identified over 200 AML-restricted targets with mesothelin (MSLN) emerged as one of the highest expressing AML-restricted targets and highly enriched in the KMT2A-rearranged AML subtype. We previously validated cell surface expression of MSLN on both AML blasts and leukemic stem cells (Le et. al. 2021). We further showed efficacy targeting MSLN in AML using antibody-drug conjugates (Kaeding et. al. 2021) and chimeric antigen receptor (CAR) T cells (Le et. al. 2021). Given that natural killer (NK) cells are potent immune effector cells and generally have a more favorable toxicity profile than CAR T cells (i.e without cytokine release syndrome), we developed CAR NK cells targeting MSLN and evaluated their efficacy in AML preclinical models. From primary patient samples, we verified MSLN cell surface expression and showed high correlation between cell surface expression (mean fluorescence intensity, MFI) and transcript expression (TPM, R = 0.72, p = 2.1x10 -8, Figure 1A) . Importantly, MSLN expression was restricted to AML blasts and entirely absent in normal lymphocytes and myeloid cells in individual patients (Figure 1B, C). Having confirmed cell surface and AML-restricted expression of MSLN, we developed CAR NK cells targeting MSLN. The VH and VL sequences from immunotoxin SS1P were used to create the single-chain variable fragment domain of the standard CAR (41-BB and CD3Zeta, Figure 1D). CAR NK cells were generated by transducing NK-92 cells with the MSLN CAR construct. Cytotoxicity of CAR NK cells was evaluated against Nomo-1 AML cell line, which expresses endogenous level of MSLN; MV4;11 and Kasumi-1 cell lines engineered to express MSLN with a lentivirus construct (MV4;11 MSLN+ and Kasumi-1 MSLN+). We initially tested the target specificity of MSLN-directed CAR NK cells against MSLN-positive (Nomo-1, MV4;11 MSLN+ and Kasumi-1 MSLN+) and MSLN-negative (Nomo-1 MSLN KO, MV4;11 and Kasumi-1) cells. CAR NK cells exhibited enhanced cytolytic activity against MSLN-positive but not MSLN-negative AML cells after 12 hours of co-incubation at the indicated effector: target (E:T) ratios (Figure 1E). We next assessed the in vivo efficacy of CAR NK cells. Nomo-1 cells transduced with GFP/Luciferase were transplanted into NSG mice at 1x10 6 cells/mouse. Unmodified or CAR NK cells were infused 1 week following leukemic cell injection at 1x10 7 cells/mouse. Monitoring leukemia burden by bioluminescence IVIS imaging showed that after 4 days post NK injection, the leukemia was significantly reduced in Nomo-1 xenograft mice treated with CAR NK cells compared to mice that received unmodified NK cells (Figure 1F), suggesting highly potent anti-leukemia activity of CAR NK cells. In this study, we demonstrate that the cell surface expression of MSLN is restricted to AML blasts but is entirely silent in normal hematopoietic subsets in individual patients. Previous and current clinical trials utilizing NK-92 cells have demonstrated safety and efficacy in variety of cancers, including AML. Here, we demonstrate that NK-92 cells genetically modified with a CAR to redirect their specificity against MSLN-positive AML cells exhibit potent, target-dependent anti-leukemia activity in vitro and in vivo. These results provide compelling data to evaluate MSLN-directed CAR NK cell therapy in clinical trials for refractory/relapsed AML, especially for high-risk KMT2A-rearranged leukemia where majority of patients express MSLN at diagnosis and relapse. Figure 1 Figure 1. Disclosures Pardo: Hematologics, Inc.: Current Employment.

The CBFA2T3-GLIS2 (CBF/GLIS) fusion is a product of a cryptic translocation exclusively seen in refractory infant AML. Lack of relevant model systems that accurately recapitulate this infant AML has limited progress. To overcome this barrier, we developed an endothelial cell (EC) co-culture system to support malignant transformation, self-renewal, and propagation of leukemia-initiating cells (LIC) in CBF/GLIS-transduced human cord blood hematopoietic stem/progenitor cells (CB HSPCs) ex vivo. Lack of recurrent cooperating mutations suggests that CBF/GLIS fusion might be sufficient for malignant transformation. To test this, we expressed the CBF/GLIS fusion or GFP control in CB HSPCs (CBF/GLIS-CB or GFP-CB) by lentiviral transduction and placed transduced cells in either EC co-culture or myeloid-promoting culture (MC). CBF/GLIS-CB cells expanded faster with prolonged lifespan in EC co-culture compared to MC (Figure 1A). Proliferation of CBF/GLIS-CB cells declined after transfer to either an EC trans-well culture or in suspension culture (Figure 1B), suggesting that direct contact as well as secreted factors are required for optimal growth of transduced cells. The CBF/GLIS fusion has been shown to confer enhanced megakaryocytic differentiation. At 6 weeks, CBF/GLIS-CB cells in EC co-culture formed significantly more megakaryocytic colonies than CBF/GLIS-CB cells grown in MC or CBF/GLIS-GFP cells grown in either condition (Figure 1C). At 12 weeks, CBF/GLIS-CB cells cultured in EC co-culture continued to produce numerous megakaryocytic colonies, demonstrating long lived self-renewal and enhance megakaryocytic differentiation of CBF/GLIS-CB cells co-cultured with ECs. To determine whether the EC niche promotes generation and propagation of LICs, we evaluated the murine engraftment of CBF/GLIS-CB cells expanded on ECs or in MC following 3, 6, 9 and 12 weeks of culture. CBF/GLIS-CB cells cultured in EC co-culture at each time point exhibited robust engraftment that progressed to frank leukemia in vivo (Figure 1D), demonstrating that EC co-culture promotes long-term maintenance of functional LICs. CBF/GLIS-CB cells grown in MC also induced leukemia from 3- and 6-week cultures but then became senescent at 9 and 12 weeks, suggesting limited preservation of the LICs. Flow cytometric analysis of CBF/GLIS-CB cells identified a malignant population that is of the RAM immunophenotype (CD56 hi, CD45 dim, and CD38 dim/-) previously reported in infants with CBF/GLIS AML in both culturing conditions. However, CBF/GLIS-CB cells in EC co-culture constituted an almost homogeneous population that expressed the RAM immunophenotype, whereas only a subset was detected in MC at week 6 (Figure 1E). To determine the fidelity of transformation to primary leukemia, we performed RNA-sequencing of CBF/GLIS-CB cells cultured with ECs or in MC. Unsurpervised clustering analysis demonstrated that the CBF/GLIS-CB cells from weeks 6 and 12 in EC co-culture clustered with primary CBF/GLIS-positive patient samples, but not CBF/GLIS-CB cells cultured in MC nor GFP controls (Figure 1F). Further transcriptome analysis revealed CBF/GLIS and HSC signature genes, previously identified to be associated with CBF/GLIS AML, were both significantly enriched in CBF/GLIS-CB cells grown in EC culture relative to MC (Figure 1G). These results suggested that the signaling pathways that are aberrantly dysregulated in primary CBF/GLIS leukemia are faithfully recapitulated in CBF/GLIS-CB cells co-cultured with ECs. Despite concerted efforts, previous attempts to model CBF/GLIS AML in murine hematopoietic cells have failed to generate overt leukemia. In this study, we demonstrate that in an EC co-culture system, the CBF/GLIS oncogenic fusion is sufficient to transform human CB HSPCs that faithfully recapitulates the morphology, transcriptome and immunophenotype of CBF/GLIS AML as well as highly aggressive leukemia in xenograft models. Furthermore, the EC co-culture system provides a tractable model system to further interrogate the mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy in CBF/GLIS AML. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

Chimeric antigen receptor (CAR) Ts have been effective in pre-B ALL, but their efficacy in AML has yet to be established. A significant barrier to effective CAR T therapy for AML is the substantial overlap of cell surface antigens expressed on AML and normal hematopoietic cells. To overcome this barrier, we profiled the transcriptome of over 3000 AML cases in children and young adults and contrasted this to normal hematopoietic tissues in search for AML-restricted targets (high expression in AML, silence in normal hematopoiesis). This led to the discovery of over 200 AML-restricted genes. Of these, Preferentially Expressed Antigen in Melanoma (PRAME) is among one of the highest expressing AML-restricted genes (Figure 1A) and, given its previous track record as a target for a variety of cancers, we selected this target for further assessment and therapeutic development in AML. However, PRAME is intracellular and therefore is inaccessible for targeting with conventional CAR T. Recently, a novel approach to target intracellular antigens was developed using TCR mimic (mTCR) antibodies, which recognize peptide/human leukocyte antigen (HLA) complexes on the tumor cell surface in a similar mode of recognition as authentic T Cell Receptors (TCRs). The Pr20 antibody was developed to recognize the PRAME ALY peptide in the context of HLA-A*02. Utilizing this Pr20 antibody, we developed a mTCR CAR T targeting PRAME and evaluated its preclinical efficacy in AML. The VL and VH sequences from Pr20 were used to construct the single-chain fragment variable domain of the 41-BB/CD3ζ CAR vector. We evaluated PRAME mTCR CAR T cells against OCI-AML-2 and THP-1 AML cell lines (PRAME +/HLA-A*02 +), K562 CML cell line (PRAME +/HLA-A*02 -) and HEK293T (293T) (PRAME -/HLA-A*02 +). Using a PE-conjugated Pr20 antibody, we confirmed that OCI-AML2 and THP-1 express PRAME ALY: HLA-A*02 but not K562 and 293T by flow cytometry (Figure 1B). As further confirmation, AML blasts in primary patient samples also stained with the Pr20 antibody (Figure 1C). For in-vivo studies, leukemia-bearing mice were treated with unmodified T or PRAME mTCR CAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse 1 week following leukemia injection. Leukemia burden was measured weekly by bioluminescence IVIS imaging. Cells were treated with 10ng/mL of IFN-γ prior to co-incubation with T cells for 16 hours. PRAME mTCR CAR T cells demonstrated potent cytolytic activity against OCI-AML2 and THP1 but not against K562 or 293T cells, following co-incubation with target cells for 24 hours (Figure 1D). Consistent with potent, target-specific reactivity against PRAME ALY: HLA-A*02 positive cells, increased levels of IFN-γ, IL-2 and TNF-α were detected in cocultures of CAR T cells with OCI-AML2 and THP1 but not with K562 and 293T cells (Figure 1D). The cytolytic activity of PRAME mTCR CAR T cells extended to primary AML specimens expressing the PRAME ALY: HLA-A*02 antigen (data not shown). In-vivo efficacy of PRAME mTCR CAR T was demonstrated in OCI-AML2 and THP-1 CDX models (Figure 1E). Treatment with CAR T cells induced leukemia clearance and significantly reduced leukemia burden in OCI-AML2 and THP-1 xenograft mice, respectively, while treatment with unmodified T cells exhibited leukemia progression (Figure 1E). The anti-leukemia activity of CAR T cells resulted in enhanced survival in OCI-AML2 (p=0.0035) and THP-1 (p=0.0047) xenografts (Figure 1F). The in-vivo activity of PRAME mTCR CAR T cells was target specific, as treatment with CAR T cells did not affect leukemia burden and survival in K562 xenograft mice (Figure 1F). Given that IFN-γ promotes PRAME presentation, we investigated whether treatment of IFN-γ would enhance cytolytic activity of PRAME mTCR CAR T cells. OCI-AML2 and THP-1 cells pretreated with IFN-γ were more sensitive to cytolysis compared to untreated controls (Figure 1G). In this study, we demonstrate the therapeutic potential of targeting PRAME with mTCR CAR T cells in AML. We show potent, target-specific reactivity of PRAME mTCR CAR T cells against PRAME ALY: HLA-A*02 positive AML cells, both in-vitro and in-vivo. We further demonstrate that the activity of PRAME mTCR CAR T cells can be enhanced with IFN-γ treatment, providing a useful strategy to increase efficacy. Thus, the results presented provide a novel approach to target PRAME with CAR T cells and compelling data to evaluate PRAME mTCR CAR T cells in AML clinical trials. Figure 1 Figure 1. Disclosures Pardo: Hematologics, Inc.: Current Employment. Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Scheinberg: Eureka Therapeutics: Current equity holder in publicly-traded company.

Introduction: Despite advances in cytotoxic and targeted therapies, recurrent disease remains the most significant obstacle to long-term survival in patients with childhood and adult leukemias. As part of our efforts to identify therapies that can be repurposed for immediate use in patients with leukemia, we interrogated a library of all available antibody-drug conjugates (ADCs) whose targets are expressed in leukemias (AML or ALL). A list of targets with available ADCs was merged with transcriptome data from over 3000 pediatric and adult leukemias (AML and ALL) to identify targets that are expressed in a large cohort of leukemias with immediate therapies for repurposing. The transcriptome data set included nearly 2000 pediatric AML cases sequenced as part of Target Pediatric AML (TpAML), 419 adult AML cases from TCGA LAML and the Beat AML program, as well as 853 ALL cases from COG and St. Jude trials. In this repurposing endeavor, CD74 emerged as the most expressed transcript in AML and ALL. CD74 encodes for a cell surface protein that associates with the class II major histocompatibility complex and is involved in the regulation of antigen presentation for immune response and B-cell differentiation. STRO-001 (Sutro Biopharma) is a CD74-directed, site-specific ADC developed for the treatment of multiple myeloma and lymphomas. Given broad expression of CD74 in leukemias, we evaluated the efficacy of STRO-001 in AML and ALL preclinical models. Methods: To characterize CD74 expression, RNA-seq data obtained from pediatric and adult AML and ALL patients was examined. Cell surface expression of CD74 was determined by flow cytometry using PE labeled anti-human CD74 antibody. The CD74-targeting ADC (STRO-001) was obtained from Sutro Biopharma.The preclinical efficacy of STRO-001 was evaluated against AML and ALL cell lines and patient samples expressing various levels of CD74 in vitro and in vivo. For in vivo studies, AML and ALL cell lines were transduced with GFP/Luciferase construct, and GFP+ cells were injected intravenously into NSG mice. Leukemia burden was measured by bioluminescence (IVIS) imaging weekly. Results: Transcriptomics analysis showed CD74 expression in a majority of adult and pediatric AML (>99% of cases) and at a much higher level compared CD33 and CD123 (targets currently developed for AML, Fig. 1A). CD74 is also broadly expressed in pediatric ALL, with a significant increase in expression observed compared to CD19 and CD22 (known targets in ALL, Fig. 1B). We confirmed that CD74 is expressed on the cell surface of AML blasts in primary patient samples (Fig. 1C) as well as AML and ALL cell lines (Fig. 1D). Given confirmation of cell surface expression of CD74, we investigated whether targeting CD74 can effectively eliminate leukemia cells. We evaluated the in vitro cytotoxicity of STRO-001 against K562 (a CML cell line that does not express CD74), AML cell lines (MV4;11 and NOMO-1), and ALL cell lines (REH1 and RS4;11) with varied CD74 expression. STRO-001 demonstrates target-specific cytotoxicity against CD74-expressing AML and ALL cell lines, but not K562 cells (Fig. 1E). STRO-001 exhibited high potency in CD74 expressing cells, with IC-50s of 41nM (MV4;11), 1.3nM (NOMO-1), 0.7nM (REH-1) and 3nM (RS4;11). In vivo studies in NSG mice transplanted with AML and ALL cell lines showed high potency. Treatment with STRO-001 at 3mg/kg once a week for 3 weeks effectively eradicated the leukemia in NOMO-1, REH-1, and RS4;11-bearing xenograft mice, while disease progression was observed in untreated control mice (Fig. 1F). We further evaluated the efficacy of STRO-001 in primary patient samples. Primary leukemia specimens from 3 patients with varied CD74 expression (Fig. 1G) were incubated with increasing concentrations of STRO-001 for 3 days. STRO-001 exhibited potent anti-leukemia activity against primary AML cells with IC-50s of 17.4nM, 12.8nM, and 4.07nM, respectively (Fig. 1H). Conclusion: Through transcriptomics profiling and validation of the cell surface expression by flow cytometry, we have identified CD74 as a viable therapeutic target for AML and ALL in children and adults. We further demonstrate that targeting CD74 with STRO-001 effectively eliminates leukemia cells both in vitro and in vivo, providing the preclinical data to compel evaluation of STRO-001 in clinical trials for childhood and adult leukemia. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Background: A rare but highly aggressive type of AML that is only seen in infants with a unique immunophenotype (RAM phenotype) is caused by cryptic CBFA2T3-GLIS2 (CBF/GLIS) fusion. This infant AML is highly refractory to conventional chemotherapy with near uniform fatality despite highly intensive and myeloablative therapy (PMID 23153540). Transcriptome profiling of CBF/GLIS AML has revealed new insights into the pathogenesis of the fusion and uncovered fusion-specific molecular biomarkers that could be used for risk stratification and to inform treatment (PMID 30592296). Studying the largest cohort of these high-risk infants, we demonstrated several alterations in gene expression and transcriptional networks in these CBF/GLIS-positive patient samples that have potential for therapeutic targeting (PMID 31719049). FOLR1, which encodes for folate receptor alpha, was highly and uniquely expressed in CBF/GLIS AML but was entirely absent in AML with other cytogenetics abnormalities and in normal hematopoietic cells. Furthermore, we recently demonstrated that forced expression of CBF/GLIS enhances the proliferation and alters differentiation in cord blood (CB) CD34+ early precursors towards megakaryocytic lineage that recapitulates acute megakaryocytic leukemia seen in infants (PMID 31719049). Of significance, we showed that FOLR1 surface expression is causally linked to CBF/GLIS-induced malignant transformation, thus making it an attractive antigen for targeted therapies against CBF/GLIS AML cells. Given that chimeric antigen receptor (CAR) T cells are extremely effective at eradicating relapsed/refractory B-ALL malignancies, we developed FOLR1-directed CAR T cells for pre-clinical evaluation in CBF/GLIS AML. Methods: We generated a FOLR1-directed CAR using anti-FOLR1 binder (Farletuzumab), IgG4 intermediate spacer and 41-BB/CD3zeta signaling domains. The pre-clinical efficacy of FOLR1 CAR T cells was evaluated against CBF/GLIS AML cell lines in vitro and in vivo. CBF/GLIS AML models include CB CD34+ cells transduced with CBF/GLIS expression construct (CBF/GLIS-CB) and WSU-AML cell line. We also engineered Kasumi-1 cell line to express FOLR1 (Kasumi-1 FOLR1+) to evaluate target specificity (Figure 1A). Results: We tested the target specificity of FOLR1-directed CAR T cells against FOLR1-positive (CBF/GLIS-CB, WSU-AML, Kasumi-1 FOLR1+) and FOLR1-negative (Kasumi-1) cells. CD8 FOLR1 CAR T cells demonstrated cytolytic activity against FOLR1 positive but not FOLR1 negative cells (Figure 1B). Furthermore, both CD8 and CD4 FOLR1 CAR T cells produced higher levels of IL-2, IFN-γ, and TNF-α and proliferated more robustly than did unmodified T cells when co-incubated with FOLR1 positive but not FOLR1 negative cells (Figure 1C). These results indicate highly specific reactivity of FOLR1 CAR T cells against AML cells expressing FOLR1. We next investigated the in vivo efficacy of FOLR1-directed CAR T cells. In CBF/GLIS-CB, WSU-AML, and Kasumi-1 FOLR1+ xenograft models, treatment with FOLR1 CAR T cells induced leukemia clearance, while disease progression occurred in all mice that received unmodified T cells (Figure 1D). Activity of FOLR1 CAR T cells in vivo was target specific, as they did not limit the leukemia progression nor extend the survival of Kasumi-1 xenografts (Figure 1D). To determine whether FOLR1 is expressed on normal HSPCs, we characterized FOLR1 expression in normal CB CD34+ samples. FOLR1 expression was entirely silent in HSPC subsets (Figure 1E). Consistent with lack of expression, no cytolytic activity was detected against HPSCs Moreover, FOLR1 CAR T cells did not affect the self-renewal and multilineage differentiation capacity of normal HSPCs as compared to unmodified control T cells (Figure 1F), whereas significant eradication of colonies were detected in the CBF/GLIS-CB cells (Figure 1G). Conclusion: In this study, we demonstrate that FOLR1 CAR T effectively eradicates CBF/GLIS AML cells without compromising normal HSPCs, providing a promising approach for the treatment of high-risk CBF/GLIS AML. Transition of this CAR T to clinical development for infant AML is underway. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Cryptic inv(16)(p13.3q24.3) leading to the CBFA2T3-GLIS2 (CBF/GLIS) oncogenic fusion is exclusively seen in infants with AML and is associated with adverse outcome. Infants with this fusion are uniformly refractory to conventional therapies and despite intensive and myeloablative therapies, virtually all patients relapse with survival less than 15% (Smith et. al. 2020). In the effort to discover actionable targets for this highly refractory leukemia, we interrogated the genome and transcriptome of nearly 3,000 AML cases, including 45 cases of CBF/GLIS AML, and contrasted this to the transcriptome in normal hematopoietic tissues. Initial computational effort to identify CBF/GLIS-specific targets focused on genes that are overexpressed in CBF/GLIS AML and silent in normal hematopoietic tissues providing us with 194 candidate genes. Filtering this library of genes based on cell surface expression and prevalence in target population narrowed the target gene list to six cell surface encoding genes (FOLR1, FRAS1, HPSE2, KLRF2, MEGF10 and PCDH19). FOLR1 encodes folate receptor alpha and given available therapeutic options with FOLR1-targeting agents, this gene was selected for further studies and therapeutic assessment of available targeted therapies. Based on RNA-seq data, FOLR1 is uniquely expressed in CBF/GLIS AML as it is absent in other AML and in normal hematopoietic cells (Figure 1A) providing an opportunity to specifically target leukemia cells while sparing normal hematopoiesis. Cell surface expression of FOLR1 on AML blasts was confirmed by flow cytometry, which further shows AML-restricted expression of FOLR1 on AML cells but not in normal hematopoietic subsets in individual CBF/GLIS patient samples (Figure 1B, C). We previously showed that forced expression of CBF/GLIS fusion transcript in cord blood hematopoietic stem/ progenitor cells (CB HSPCs) induces malignant transformation that fully recapitulates primary CBF/GLIS AML (Hylkema et. al. 2019 ASH). We also demonstrated that CBF/GLIS-transduced CB HSPCs upregulate FOLR1 expression indicating that FOLR1 expression is causally associated with CBF/GLIS expression (Figure 1D). Given cell surface expression of FOLR1 on CBF/GLIS AML cells, we next investigated the preclinical efficacy of targeting FOLR1 using FOLR1-directed site-specific antibody-drug conjugate (STRO-002, Sutro Biopharma). In vitro efficacy of STRO-002 was tested against MV4;11 AML cell line engineered to express FOLR1 (MV4;11 FOLR1+, Figure 1D) and CBF/GLIS-transduced CB HPSCs. STRO-002 exhibited high cytotoxicity against MV4;11 FOLR1+ and CBF/GLIS-transduced CB HPSCs with IC50s of 0.1nM and 4.2nM, respectively (Figure 1E). In vivo efficacy of STRO-002 was evaluated in MV4;11 FOLR1+ NSG xenograft models, which showed potent activity that led to complete leukemia clearance after 3 weekly doses of STRO-002 at 2.5 and 5 mg/kg (Figure 1F). The anti-leukemia effects of STRO-002 resulted in significant increase in survival (p=0.002, Figure 1G). STRO-002 showed similar in vivo efficacy in the xenograft mice bearing CBF/GLIS-transduced CB HSPCs (Figure 1H) suggesting highly potent activity of STRO-002 against FOLR1+ AML cells. In this study, we utilize a computational approach to discover AML-restricted targets (high expression in AML, silent in normal) that are previously elusive to identify actionable targets for high-risk CBF/GLIS AML. From this discovery pipeline, we demonstrate FOLR1 to be highly and uniquely expressed in CBF/GLIS AML but not in normal counterparts, providing a strategy to target leukemia cells without impacting normal hematopoiesis. Finally, we demonstrate that targeting FOLR1 with STRO-002 antibody drug conjugate effectively eliminates CBF/GLIS-positive AML in vitro and in vivo, providing a promising approach to eradicate leukemia and improve outcomes in this high-risk AML subtype. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

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.

Mutations affecting DNA polymerase exonuclease domains or mismatch repair (MMR) generate “mutator” phenotypes capable of driving tumorigenesis. Cancers with both defects exhibit an explosive increase in mutation burden that appears to reach a threshold, consistent with selection acting against further mutation accumulation. In Saccharomyces cerevisiae haploid yeast, simultaneous defects in polymerase proofreading and MMR select for “antimutator” mutants that suppress the mutator phenotype. We report here that spontaneous polyploids also escape this “error-induced extinction” and routinely outcompete antimutators in evolved haploid cultures. We performed similar experiments to explore how diploid yeast adapt to the mutator phenotype. We first evolved cells with homozygous mutations affecting polymerase δ proofreading and MMR, which we anticipated would favor tetraploid emergence. While tetraploids arose with a low frequency, in most cultures, a single antimutator clone rose to prominence carrying biallelic mutations affecting the polymerase mutator alleles. Variation in mutation rate between subclones from the same culture suggests that there exists continued selection pressure for additional antimutator alleles. We then evolved diploid yeast modeling MMR-deficient cancers with the most common heterozygous exonuclease domain mutation (POLE-P286R). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the POLE-P286R homologous allele (pol2-P301R) that suppressed the mutator phenotype as much as 100-fold. Independent adaptive events in the same culture were common. Our findings suggest that analogous tumor cell populations may adapt to the threat of extinction by polyclonal mutations that neutralize the POLE mutator allele and preserve intratumoral genetic diversity for future adaptation.

DNA polymerase (Pol) proofreading and mismatch repair (MMR) defects produce “mutator” phenotypes capable of driving tumorigenesis. “Ultra-mutated” cancers with defects in both pathways exhibit an explosive increase in point mutation burden that appears to reach an upper limit of ∼250 mutations/Mb, which may be evidence for negative selection. Simultaneous defects in proofreading and MMR drive error-induced extinction ( EEX ) of haploid yeast. Some yeast mutants escape extinction through antimutator mutations that suppress the mutator phenotype. We report here that other yeast mutants escape by spontaneous polyploidization. We sought to compare these two adaptive strategies during the evolution of haploid and diploid mutators. We first evolved 89 independently isolated haploid mutator strains with mutation rates an order of magnitude below the lethal threshold. Polyploid cells overtook nearly every culture. In a similar manner, we evolved strong diploid mutators, but found only a single tetraploid emerged after ∼250 cellular generations. Antimutators dominated these late passage cultures and were present after only 30-40 cellular generations. Whole genome sequencing of independent isolates from the same culture allowed us to construct phylogenetic trees that revealed that a single subclone swept through each culture carrying multiple antimutator mutations affecting the mutator polymerase. Variation in mutation rate between subclones from the same culture suggests some strains carry additional antimutator alleles in other genes. Our findings in diploid mutator yeast suggest that mutator-driven cancer cells may also adapt to the threat of extinction by accumulating multiple antimutator alleles that cooperatively reduce mutation rate to tolerable levels. Author summary “Mutator” tumor cells that cannot correct DNA replication errors exhibit an extremely high mutation rate that accelerates their evolution. But this gamble puts them at risk for extinctiondue to the accumulation of harmful mutations. Mutator yeast cells escape extinction through “antimutator” mutations that lower mutation rate. We discovered that yeast cells also adapt by duplicating their genome. To understand which adaptation may emerge in mutator-driven tumors, we evolved haploid and diploid mutator yeast cells by long-term propagation. Haploid mutators primarily adapted by genome duplication, thereby protecting themselves from recessive lethal mutations. In contrast, diploid mutators evolved by acquiring multiple antimutator mutations. Our results suggest that mutator-driven tumors may similarly accumulate combinations of mutations that attenuate their high mutation rates.

Background: Acute myeloid leukemia (AML) is one of the most highly refractory hematologic malignancies despite intensive combination chemotherapy and bone marrow stem cell transplantation. Lack of curative treatments is in large part due to our poor understanding of the disease biology and paucity of therapeutic targets. In an effort to identify actionable targets, we recently completed the largest genome, epigenome and transcriptome profiling of AML in nearly 3000 children and young adults. This discovery effort has led to the identification of a library of novel AML-restricted targets (high expression in AML, minimal-to-no expression in normal hematopoiesis) for therapeutic development. One such target was MSLN which encodes for mesothelin, a cell surface adhesion molecule that is highly expressed in 30-50% of AML cases in pediatric (Children Oncology Group) and adult (MD Anderson) cohorts and is entirely absent in normal bone marrow and peripheral blood CD34+ cells. MSLN expression in normal tissues is confined to mesothelial cells lining the pleura, pericardium, and peritoneum. Previous studies targeting MSLN in solid tumors have demonstrated clinical efficacy with minimal toxicities. Given that T cells genetically modified to express chimeric antigen receptors (CARs) are extremely effective at eradicating relapsed and refractory malignancy, we developed MSLN-directed CAR T cells for pre-clinical evaluation in AML. Methods: From primary patient samples, we verified MSLN expression by RT-PCR and confirmed mesothelin surface protein expression on leukemic blasts by flow-cytometry as well as detected soluble mesothelin in the plasma by ELISA. The VH and VL sequences from Amatuximab were used to create the scFv domain of the standard CAR (41-BB and CD3Zeta). For in vivo CAR T study, Nomo-1 cells, which express endogenous level of MSLN, and Kasumi-1 cells engineered to express MSLN with a lentivirus construct (Kasumi-1 MSLN+) were transplanted into NSG mice. Mock transduced MSLN-directed CAR T cells were infused 1 week (Nomo-1) and 2 weeks (Kasumi-1 MSLN+) following leukemic cell injection. Leukemic burden was measured by bioluminescence IVIS imaging weekly. For in vitro study, Nomo-1 cells were treated with GM6001 (50uM), a metalloprotease inhibitor, or DMSO control for 48 hr prior to evaluation of surface mesothelin by flow cytometry and soluble mesothelin in the culture supernatant by ELISA. Results: In vivo cytotoxicity of CAR T cells against Nomo-1 and Kasumi-1MSLN+ AML models demonstrated potent, target-dependent tumor killing. After 1- and 2-weeks post CAR T infusion, leukemic cells were eradicated in both Nomo-1 (p Conclusion: In this study, we demonstrate that mesothelin is a viable therapeutic target and a potential diagnostic biomarker in AML. We show that MSLN CAR T cells were highly effective in eliminating MSLN-positive AML cells in vitro and in vivo. Shedding contributes to the loss of mesothelin antigen and provides a source of soluble mesothelin that may interfere with antibody-based therapies, including CAR T cells. Modulating MSLN shedding by inhibiting ADAM17-mediated cleavage resulted in stabilized mesothelin and improved CAR T cell functionality. This work warrants further evaluation of MSLN CAR T cells to be tested in clinical trials for AML and demonstrates that inhibiting MSLN shedding is a promising approach to improve CAR T efficacy. Disclosures No relevant conflicts of interest to declare.