Abstract: Chimeric Antigen Receptor (CAR) therapy has led to great successes in patients with leukemia and lymphoma. Umbilical Cord Blood (UCB), stored in UCB banks, is an attractive source of T cells for CAR-T production. We used a third generation CD123 CAR-T (CD28/4-1BB), which was previously developed using an adult’s Peripheral Blood (PB), to test the ability of obtaining CD123 CAR-T from fresh or cryopreserved UCB. We obtained a cell product with a high and stable transduction efficacy, and a poorly differentiated phenotype of CAR-T cells, while retaining high cytotoxic functions in vitro and in vivo. Moreover, CAR-T produced from cryopreserved UCB are as functional as CAR-T produced from fresh UCB. Overall, these data pave the way for the clinical development of UCB-derived CAR-T. UCB CAR-T could be transferred in an autologous manner (after an UCB transplant) to reduce post-transplant relapses, or in an allogeneic setting, thanks to fewer HLA restrictions which ease the requirements for a match between the donor and recipient.

Abstract: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive acute leukemia/lymphoma recently classified as a malignant transformation of plasmacytoid dendritic cells (pDCs) and a subtype of acute myeloid leukemia (AML). BPDCN has no standard treatment and a poor prognosis, with median survival 〈 1 year. A significant roadblock to better understanding BPDCN is a lack of adequate model systems. We generated patient-derived xenografts (PDX) of BPDCN in NOD/Scid/IL2rgnull (NSG) mice. Bone marrow or peripheral blood cells involved by BPDCN blasts (CD45 low, CD123 high, HLA-DR high, CD3 neg) were transplanted into irradiated NSG recipients. Nine of 16 BPDCNs caused lethal leukemia involving blood, spleen, and bone marrow 2-6 months after transplantation. All nine BPDCN PDXs were serially transplantable. Flow characterization of each patient's BPDCN and corresponding xenograft revealed no major differences in BDCA2, BDCA4, FCeR1, ILT7, or cytoplasmic TCL1 staining. All samples maintained high expression of the human interleukin-3 (IL3) receptor (IL3Ralpha/CD123), a hallmark feature of BPDCN. To further characterize BPDCN pathogenesis we performed whole transcriptome sequencing (RNA-seq) on sorted blasts from 11 patients and on normal pDCs isolated from 4 healthy donors. These were compared to RNA-seq in six PDXs. The spectrum of mutations in BPDCN transcriptomes overlapped with that seen in other hematologic malignancies, particularly myeloid disorders, and was similar to reported DNA mutations in BPDCN, including in ASXL1, CTCF, IDH2, NRAS, RUNX1, STAG2, TET2, and TP53. Particularly striking was the presence of a canonical mutation in an RNA splicing factor in 7 of 11 cases (SRSF2 P95H/L/R in four, ZRSR2 R295* and gene locus deletion in two, and SF3B1 K666N in one). Known oncogenic mutations in the original disease were retained in the PDXs, including all splicing factor mutations, with the exception of an IDH2 R140Q that was lost in one PDX. BPDCN PDXs grouped together in unsupervised clustering of expression profiles, distinct from AML and ALL PDXs in an analysis of 134 models from the DFCI Public Repository of Xenografts (http://PRoXe.org). Gene set enrichment analysis (GSEA) of KEGG and REACTOME pathways associated with differentially expressed genes between primary BPDCNs and non-malignant pDCs revealed signatures related to dendritic cell activation, cell cycle, and apoptosis. In addition, 3 of the top 11 sets were genes involved in mRNA processing, mRNA splicing, and processing capped intron-containing pre-mRNAs (all FDR 〈 1e-6). To test the efficacy of BPDCN-targeted therapy using primary human leukemias in vivo, we performed a pre-clinical trial in NSG mice using SL-401, a recombinant biologic consisting of a fusion protein of IL3 and diphtheria toxin. Three independent BPDCN xenografts were injected into 20 NSG mice each, and followed by weekly peripheral blood monitoring for human CD45 and CD123. When leukemia burden reached 〉 0.5% in at least half of the mice in the cohort, animals were randomized to receive SL-401 at 100 ug/kg or vehicle intraperitoneally daily for 5 days. Two mice in each group were sacrificed at day 7 for response assessment, and peripheral blood was followed weekly in the remaining mice for evidence of progression ( 〉 5% human CD45/CD123-positive cells). 7 days after treatment, mice receiving SL-401 had dramatic reductions in BPDCN in the peripheral blood, spleen, and bone marrow (0.31% vs 37.6% in marrow of SL-401 vs vehicle). SL-401 prolonged progression-free survival in all BPDCNs tested (12 vs 48 days, P 〈 0.0001 by log-rank test). At the time of progression after SL-401, relapsing mice were re-randomized to receive a 2nd and in some cases 3rd cycle of SL-401 or vehicle. Repeated treatment in mice that progressed after SL-401 resulted in second and third peripheral blood remissions. All PDXs responded to SL-401 including those with and without splicing factor and TP53 mutations. CD123 expression was maintained at high levels on all SL-401 treated BPDCNs even after repeated cycles. Primary xenografts of BPDCN are faithful models of the human disease, maintain genetic and transcriptomic characteristics of the original tumor, and respond to multiple courses of IL3-DT in vivo, suggesting that they provide a valuable resource to study disease biology and response/resistance to targeted therapy. Disclosures Chen: Stemline Therapeutics, Inc.: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lane:Stemline Therapeutics, Inc.: Research Funding.

Abstract: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, male-biased ( & gt;3:1 M:F) hematologic malignancy in which some patients have bone marrow involvement at diagnosis (50%) and most have tumor formation in the skin (~90%), often preceding marrow disease. The prognosis is poor (median survival of 12-24 months) and there is unmet need for biological insight. TET2, ASXL1, and RNA splicing genes (SRSF2, SF3B1, and ZRSR2) are recurrently mutated in BPDCN. The X chromosome gene ZRSR2 was the most frequently mutated spliceosome gene reported in a prior BPDCN cohort (7 of 24, 29.2%; Taylor, ASH 2013). Our goal was to define the functional consequences of ZRSR2 mutations in BPDCN. First, we confirmed the frequency of ZRSR2 mutations in a larger cohort from the US and Europe; we found ZRSR2 mutations in 24 of 93 (25.8%). Notably, ZRSR2 mutations were almost exclusively in males (23/73 males vs 1/20 females, P=0.019). Next, we compared the global mutation pattern to 30 predefined signatures from & gt;7000 cancers in COSMIC. Analysis of all somatic single nucleotide variants in 11 tumor-normal pairs using whole exome sequencing (tumor was sorted BPDCN cells from marrow) revealed that BPDCN had an ultraviolet (UV)-induced mutation signature (score & gt;0.25 in 6/11 or 55%; Figure 1A). For comparison, we detected the UV signature in melanoma but not in AML from The Cancer Genome Atlas. These data suggest that mutations acquired in the skin stage of BPDCN evolution are retained in subsequent leukemic disease. Next, we performed RNA-sequencing from sorted BPDCN and normal plasmacytoid dendritic cells (pDCs). Differentially expressed genes between BPDCN and pDCs (BCL2, MYB, IRF4, CEP70, IGLL1, GZMB) were similar to those that distinguish BPDCNs from pDCs by bulk and single cell RNA-sequencing. By gene set enrichment analysis (GSEA), BPDCNs were enriched for overexpression of MYC/E2F targets and PI3K/AKT/MTORC1 signaling pathway-associated genes. BPDCNs transcriptomes were also enriched for gene sets associated with RNA splicing machinery and RNA nonsense mediated decay (NMD). To link RNA splicing with functional consequences of ZRSR2 mutations, we generated ZRSR2-knockout BPDCN cells (CAL1) using CRISPR/Cas9. This models primary tumors because ZRSR2-mutant BPDCNs have complete loss of ZRSR2 protein. Activation marker (CD80) upregulation and type 1 interferon secretion after Toll-like receptor (TLR) stimulation with lipopolysaccharide (LPS) or R848 were reduced in ZRSR2-deficient cells. We found similar defective cytokine production in stimulated primary BPDCN cells compared to normal pDCs. After activation, normal pDCs undergo apoptosis in a negative feedback process. In contrast, ZRSR2-knockout, but not control cells, were protected from TLR activation-induced apoptosis. Reexpression of wild-type ZRSR2 in knockout cells restored activation-induced apoptosis (Figure 1B). These data suggested that ZRSR2-mutant BPDCNs have defects downstream of TLR stimulation. By RNA-sequencing, we found that IRF7 mRNA was mis-spliced in all ZRSR2- (2/2), SRSF2- (4/4), and SF3B1- (1/1) mutant BPDCNs compared to those with no mutated splicing gene (4/4). IRF7 (interferon regulatory factor 7) is a transcription factor activated by TLR signaling that is important for pDC activation and apoptosis. The IRF7 mRNA transcript contains a "weak intron" (intron 4) that is subject to intron retention, which leads to NMD and reduced IRF7 protein level in stimulated dendritic cells (Luke, Mol Cell 2019). IRF7 intron 4 was mis-spliced in ZRSR2-, SRSF2-, and SF3B1-mutant BPDCNs. ZRSR2-knockout CAL1 cells had severely impaired ability to upregulate IRF7 after LPS stimulation, which was partially rescued by reepxression of wild-type ZRSR2 (Figure 1C). Expression of constitutively activated IRF7 inhibited growth of both ZRSR2-knockout and control cells, confirming that the inability to activate IRF7 is important for the effect of ZRSR2 loss on TLR agonist-induced growth inhibition. In conclusion, male-biased ZRSR2 mutations are frequent in BPDCN and impair pDC activation and apoptosis, at least in part via TLR-IRF7. These data may explain why BPDCNs have an impaired activation state (Bierd, BCJ 2019). They also suggest that splicing factor mutations affect cell type-specific pathways to promote transformation, underscoring the importance of studying cancer genes in relevant contexts. Figure Disclosures Griffin: Moderna Therapeutics: Consultancy. Ghandi:Monte Rosa Therapeutics: Consultancy; Cambridge Data Science LLC: Current Employment, Current equity holder in private company. Seiler:Remix Therapeutics: Current Employment. Konopleva:Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; Agios: Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding; Calithera: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Pemmaraju:Cellectis: Research Funding; Daiichi Sankyo: Research Funding; DAVA Oncology: Honoraria; Plexxikon: Research Funding; Blueprint Medicines: Honoraria; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support; Celgene: Honoraria; Pacylex Pharmaceuticals: Consultancy; Affymetrix: Other: Grant Support, Research Funding; MustangBio: Honoraria; Roche Diagnostics: Honoraria; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Samus Therapeutics: Research Funding. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company. Lane:Qiagen: Consultancy; Abbvie: Research Funding; Stemline Therapeutics: Research Funding.

Abstract: A new entity of acute leukemia co-expressing CD4 and CD56 markers without any other lineage-specific markers has recently been identified as arising from plasmacytoid dendritic cells (pDC). We proposed to call it plasmacytoid dendritic cell leukemia (pDCL) (Chaperot et al., 2001; Feuillard et al., 2002). We previously reported that pDCL may express the CD33 myeloid associated marker (Garnache-Ottou et al., 2005). This expression of CD33 on CD4+CD56+ lineage negative cells should not exclude the diagnosis of pDCL and underlines that pDCL specific markers should be identified. In order to better characterize pDCL, we organized a French pDCL network to collect cells and data on this rare leukemia. The aim of this study was to identify specific markers for the routine diagnosis. We focused on previously described pDC specific markers: CD123, BDCA-2, BDCA-4 (neuropilin-1). Fourteen pDCL and 69 other acute leukemias (10 B-ALL, 3 T-ALL, 3 BAL, 2 unclassified AML, 4 AML M0, 12 AML M1, 11 AML M2, 1 AML M3, 11 AML M4, 9 AML M5 according to FAB classification and 3 blastic transformations of myeloproliferative or myelodysplastic syndromes) were analyzed by flow cytometry. Leukemic cells were identified on their low expression of CD45 and using lineage specific markers. pDCL have been functionally characterized by their capacities to activate naïve cord blood CD4+ T cells, to induce a Th2 polarization and to produce IFN-α in response to viral supernatant. BDCA-4 was not specific for pDCL since 10% of acute leukemias (7/69) expressed this marker at the same levels as pDCL. This was not surprising since BDCA-4 has been reported to be expressed by myeloid cells as well as by some B and T cell subsets. Unlike BDCA-4, BDCA-2 was never expressed in the 69 tested acute leukemias. However, BDCA-2 expression was not systemically detected in all the pDCL tested (4 negative cases out of the 14 tested). CD123, also know as IL-3 receptor α-chain, was expressed on nearly all the acute leukemias tested. However, the levels of expression discriminated pDCL from other acute leukemias. These latter acute leukemias expressed lower levels of CD123 (fluorescence intensity ratio: 22 [mean] +/- 8 [SEM] ; range: 2–71) when compared to pDCL (fluorescence intensity ratio: 171 [mean] +/− 36 [SEM] ; range: 71–353). Overall, these results show that BDCA-4 is not a useful marker to identify pDCL. When BDCA-2 is expressed on leukemic cells, this is a strong argument in favor of pDCL. Finally, the level of CD123 expression is an interesting tool to characterize pDCL. We plan to analyze other acute leukemias, and in particular, acute leukemias aberrantly expressing CD4 and/or CD56 markers. This will allow us to confirm that pDCL can be identified on the basis of BDCA-2 and CD123 expression. Since the prognosis of pDCL was poor, an early diagnosis is needed to treat patients with specific therapeutic options (e.g., allogeneic hematopoietic cell transplantation). This can be achieved in the future by using anti-BDCA-2 and CD123 antibodies in addition to classical lineage specific markers. supported by the Goelam and the GEIL

Abstract: Blastic plasmacytoid dendritic cell neoplasm is a clonal disease derived from precursors of plasmacytoid dendritic cells (pDC). It is a rare neoplasm involving the skin which may or may not be associated from the outset with a leukemic component. The disease invariably progresses to aggressive leukemic dissemination, leading to a differential diagnosis with acute leukemia. In 2004, we set up a French network to recruit biological data at diagnosis. Diagnosis was according to recommendations (Swerdlow et al, 2008), with, in addition, a mandatory panel of pDC markers (Garnache-Ottou et al, 2009) detected by flow cytometry or by immunohistochemistry on infiltrated blood, bone marrow or cutaneous lesions. In total, 109 cases of BPDCN were included in 35 hospitals (2000-2013). BPDCN is more prevalent in men (sex ratio 4.4/1) and in elderly subjects (median age: 63 years; 7 patients were 〈 20 yo).S kin lesions are very prevalent (85%) with variable lesion types. Blood cell counts show variable leukocytosis (figure 1A) with presence of blasts in 65% of cases. Anemia and thrombopenia were present in 59% and 76% of cases respectively. Bone marrow aspiration showed blastic infiltration in 94% of cases. Indeed, in 7 cases, there was isolated cutaneous involvement at diagnosis, with neither blood nor bone marrow infiltration. Morphologies of blast cells were heterogeneous. Typical morphologies were the most frequent, including medium-sized cells with a blastic round or irregular nucleus, cytoplasm displayed faint and irregular basophilia and no granulation. In a contingent of the blastic populations, we observed small vacuoles in a peculiar arrangement under the cytoplasmic membrane (42%) or the presence of large pseudopodia (28%) or both (17%) (Figure 1B, C, D). Some cases showed a more immature morphology with larger cells, higher nucleo-cytoplasmic ratio, very visible nucleoli and reinforced basophilia (28%) or a pseudomonoblastic morphology (5%) (Figure 1E). Rare cases presented a pseudolymphocytic form (7%, figure 1F) or large granulations in the cytoplasm (2%). Peroxidase and esterase were negative in all cases. Dysplasia of hematopoietic lineages was observed in 29% (figure 1G). For 8% of patients, myelodysplastic syndrome was diagnosed before the diagnosis of BPDCN. Immunophenotype showed that HLA-DR and CD4 were expressed in all cases, but 4 cases did not express CD56 (confirmed using 3 different antibodies). Expression of markers of others hematopoietic lineages was frequent. Among myeloid markers, the most frequent was CD33 (46%), followed by CD117 (23%), whereas CD13, CD11c, CD15 and CD65 were rarely expressed. Monocyte markers (CD14, CD64, CD11b) and myeloperoxidase were never expressed. For the T lineage, CD2 and CD7 were the most frequent (62% and 58% respectively) whereas CD5 was rare (7%). No cytoplasmic or surface CD3 were detected. For the B lineage, CD22 was expressed in 16%, and low levels of cCD79a in 5%. Both were never expressed together, and no CD19, CD20 and immunoglobulins were found. Generally, we observed one of these antigens (Ags) per case, but in 44% of cases, there was a combination of 2 or 3 Ags from 2 or 3 different lineages. Immature Ags such as CD34 and CD133 were never found, and Tdt was found in 14% of cases. Cytogenetic analysis revealed abnormal caryotype in 65% of the 78 caryotypes evaluated, with 20 cases having a complex caryotype. The frequency of the chromosomal abnormalities involved are shown in Figure 1H. In conclusion, we describe the largest series of BPDCN to date in the literature. Detailed clinical and biological data at presentation allow improved recognition of this rare form of acute and aggressive leukemia, enabling early initiation of appropriate management. Figure 1. A: Blood cell count in 109 BPDCN patients at diagnosis. Bars represent the median. B: Typical BPDCN morphology. C: In this case, the nuclei were peripheral, cytoplasm presented heterogenous basophilia, vacuoles were rare but large pseudopodia are frequent. D: Typical morphology with frequent microvacuoles under the cytoplasmic membrane. E: Immature morphology. F: Pseudolymphocytic morphology. G: Presence of dysplasia in myeloid cells with Auer Rods in the granulocytes. The morphology of the Blastic cells is typical. H. Chromosomal abnormalities in 78 caryotypes evaluated: The histogram represents the number of cases in which each chromosome was involved (deletion, gain, translocations). Figure 1. A: Blood cell count in 109 BPDCN patients at diagnosis. Bars represent the median. B: Typical BPDCN morphology. C: In this case, the nuclei were peripheral, cytoplasm presented heterogenous basophilia, vacuoles were rare but large pseudopodia are frequent. D: Typical morphology with frequent microvacuoles under the cytoplasmic membrane. E: Immature morphology. F: Pseudolymphocytic morphology. G: Presence of dysplasia in myeloid cells with Auer Rods in the granulocytes. The morphology of the Blastic cells is typical. H. Chromosomal abnormalities in 78 caryotypes evaluated: The histogram represents the number of cases in which each chromosome was involved (deletion, gain, translocations). Disclosures Bardet: Celgene: Research Funding. Deconinck:CHUGAI: Other: Travel for international congress; PFIZER: Research Funding; ROCHE: Research Funding; NOVARTIS: Other: Travel for international congress; ALEXION: Other: Travel for international congress; JANSSEN: Other: Travel for international congress; LFB loboratory: Consultancy.

Abstract: Abstract 2588 Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive neoplasm derived from plasmacytoid dendritic cell precursors that involves the skin and invariably leads to aggressive leukemic dissemination (Garnache Ottou et al., 2007). The incidence is more frequent in men and elderly subjects, but BPDCN may occur in young adults and even in children. Current treatment options are not codified, and although conventional chemotherapy often induces an initial response, relapse is frequent and rapid. The prognosis is poor, with a median overall survival of 9–13 months whatever the initial presentation was. Allogeneic hematopoietic transplantation is currently the only potentially curative treatment, but this is not an option for most elderly patients. Therefore, there is an urgent need to develop novel therapies that can specifically target this tumor type. Since BPDCN cells express high levels of the interleukin-3 receptor (IL-3R) α-chain (CD123), we tested SL-401, a novel biologic conjugate that targets IL-3Rα (Konopleva et al. 2010 and Frolova et al. 2010), against 2 BPDCN cell lines (GEN2.2 and CAL-1) and 6 primary BPDCN cells isolated from 5 patients (P#1-5). Cells from P#2 were tested at diagnosis (#2d) and at relapse (#2r). A CD123+ cell line (TF-1/H-ras) sensitive to SL-401 and a CD123− cell line (Daudi) were used as controls. Cytotoxicity was assessed by MTT assay as well as flow cytometry (FC) after Annexin-V (AV) and 7-AAD staining. Primary BPDCN cells were cultured with IL-3 alone (to maintain survival) or IL-3 in combination with SL-401 for 24 h (FC) or 48 h (MTT) (Figure 1). All BPDCN cell lines and primary cells were found to be sensitive to SL-401 by MTT assay (Figure 1A). These results were confirmed by FC with a dose-dependent decrease in cell viability observed for the GEN2.2 and CAL-1 lines, (47 ± 10 to 2 ± 2%, n = 4, and 90 ± 7 to 11 ± 5%, n = 4, respectively) when treated with SL-401 compared to untreated cells (48 ± 9% and 88 ± 5%, for GEN2.2 and CAL-1 respectively). Cell viability decreased for all the 6 primary cells tested by FC (Figure 1B). The Daudi negative control cell line was resistant to SL-401 (Figure 1A), which confirmed SL-401 specificity.Figure 1.In vitro sensitivity of BPDCN cell lines (CAL-1 and GEN2.2) and primary cells from 5 BPDCN patients to SL-401 assessed by FC and MTT assays.Figure 1. In vitro sensitivity of BPDCN cell lines (CAL-1 and GEN2.2) and primary cells from 5 BPDCN patients to SL-401 assessed by FC and MTT assays. This is the first study evaluating the in vitro sensitivity of BPDCN using the IL-3R targeted drug candidate, SL-401, which is currently being evaluated in clinical trials of patients with acute myeloid leukemia (AML), myelodysplastic syndrome, and chronic myeloid leukemia. Importantly, the highest concentration of SL-401 evaluated in these in vitro studies was ten times lower than the peak plasma concentration achieved in AML patients (Frankel et al, 2008). Since all BPDCN patients evaluated to date express high levels of CD123 (Garnache Ottou et al, 2009), these results suggest that BPDCN patients may clinically benefit from SL-401 therapy. This new strategy should be evaluated in a clinical trial. A. Percentage of viable cells from BPDCN patients (#1, #4, and #5, n = 1) or of viable CAL-1 and GEN 2.2 cells (n = 3) assessed by MTT assay after incubation with different concentrations of SL-401 or without drug (cells only). Untreated cells were considered as 100% viable cells. The Daudi cell line (CD123−) was used as a negative control (n = 3). B. Percentage of viable cells (AV and 7-AAD negative, as measured by FC) after incubation with different concentrations of SL-401 or without drug (cells only) for 24 h. The histogram represents a mean of 3 (P#1), 4 (P#2d), and 6 (P#2r) independent experiments. Samples (P#3- 4, and- 5) were each tested once. Disclosures: Frankel: Stemline Therapeutics: Patents & Royalties, Research Funding. Jacobson:Stemline Therapeutics, Inc: Employment, stock Options. Cirrito:Stemline Therapeutics, Inc: Employment, Equity Ownership, Patents & Royalties. Brooks:Stemline Therapeutics, Inc: Employment, equity options.

Abstract: Chronic myeloid leukemia (CML) is a chronic disease resulting in myeloid cell expansion through expression of the BCR-ABL1 fusion transcript. Tyrosine kinase inhibitors (TKI) have significantly increased survival of patients with CML, and deep responders may consider stopping the treatment. However, more than 50% of patients relapse and restart TKI, subsequently suffering unknown toxicity. Because CML is a model immune system–sensitive disease, we hypothesize that chimeric antigen receptor (CAR) T cells targeting IL1 receptor-associated protein (IL1RAP) in quiescent CML stem cells may offer an opportunity for a permanent cure. In this study, we produced and molecularly characterized a specific monoclonal anti-IL1RAP antibody from which fragment antigen-binding nucleotide coding sequences were cloned as a single chain into a lentiviral backbone and secured with the suicide gene iCASP9/rimiducid system. Our CAR T-cell therapy exhibited cytotoxicity against both leukemic stem cells and, to a lesser extent, monocytes expressing IL1RAP, with no apparent effect on the hematopoietic system, including CD34+ stem cells. This suggests IL1RAP as a tumor-associated antigen for immunotherapy cell targeting. IL1RAP CAR T cells were activated in the presence of IL1RAP+ cell lines or primary CML cells, resulting in secretion of proinflammatory cytokines and specifically killing in vitro and in a xenograft murine model. Overall, we demonstrate the proof of concept of a CAR T-cell immunotherapy approach in the context of CML that is applicable for young patients and primary TKI-resistant, intolerant, or allograft candidate patients. Significance: These findings present the first characterization and proof of concept of a chimeric antigen receptor directed against IL1RAP expressed by leukemic stem cells in the context of CML.