Human Acute Myeloid Leukemia Blasts Research Articles

Selective degradation of mutant FMS-like tyrosine kinase-3 requires BIM-dependent depletion of heat shock proteins

AbstractInternal tandem duplications in the FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). Proteolysis-targeting chimeras (PROTACs) that induce proteasomal degradation of mutated FLT3 emerge as innovative pharmacological approach. Molecular mechanisms that control targeted proteolysis beyond the ubiquitin-proteasome-system are undefined and PROTACs are the only known type of FLT3 degraders. We report that the von-Hippel-Lindau ubiquitin-ligase based FLT3 PROTAC MA49 (melotinib-49) and the FLT3 hydrophobic tagging molecule MA50 (halotinib-50) reduce endoplasmic reticulum-associated, oncogenic FLT3-ITD but spare FLT3. Nanomolar doses of MA49 and MA50 induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD (p < 0.05-0.0001), but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells, and c-KIT-dependent cells. In vivo activity of MA49 against FLT3-ITD-positive leukemia cells is verified in a Danio rerio model. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein-folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced AML patient survival (p < 0.1) and HSP90, HSP110, and BIM are linked to the expression of FLT3 in primary AML cells (p < 0.01). HSP90 suppresses degrader-induced FLT3-ITD elimination and thereby establishes a mechanistically defined feed-back circuit.

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells.

Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient's death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient's bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.

Adaptor Anti-P329G CAR T Cells for Modular Targeting of AML

Introduction Chimeric antigen receptor (CAR) T cell therapy has achieved promising results for treatment of B cell and plasma cell malignancies, but success has been limited in the treatment of acute myeloid leukemia (AML). Numerous antigens including CD33, CD123 or CSF1R have been proposed for AML therapy. However, clinical success of CAR T cell therapy in AML is impaired by antigen heterogeneity, therapy-associated toxicities, and antigen escape. Adaptor CAR T cells which allow sequential, transient or simultaneous targeting of multiple antigens have the potential to overcome these limitations. These adaptor CAR T cells were combined with tumor antigen-directed CAR-adaptor molecules for indirect tumor binding. We developed a modular P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human IgG1 antibodies containing P329G L234A/L235A (PGLALA) mutations. These PGLALA mutations render the Fc part inactive for binding to Fc gamma receptors (FcgRs) and to the complement system, and up to now, more than 20 such Fc-silenced therapeutic antibodies including an approved T cell recruiting bispecific antibody have been clinically validated. Compared to other modular CAR T cell platforms this approach does not rely on haptens or artificial tags fused to the targeting antibody and may ease development and testing of the platform. Methods A scFv-based second generation CAR vector system was used in primary human T cells. P329G-targeting CAR T cells were combined with PGLALA-Fc-mutated antibodies targeting CD33, CD123 or CSF1R. Classical, directly binding anti-CD33 and anti-CSF1R CAR T cells have been used as positive controls, while anti-CD19 CAR T cells and untransduced T cells have been used as negative controls. Different AML (MOLM-13, OCI-AML-3, PL-21, Mv4-11, THP-1 and U-937) and an ALL cell line (NALM-6) were used as target cell lines expressing the target antigens in different intensity. Effector function of CAR T cells was also tested against primary human AML blasts derived from patients. Luciferase-based killing assays, ELISA-based and flow-based analysis were used to evaluate CAR effector functions. Results Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, anti-P329G CAR T cells revealed specific effector functions only when combined with antibodies carrying PGLALA-Fc-mutations. Anti-P329G CAR T cells alone revealed no activation. Anti-P329G CAR T cells could be specifically activated by recombinant protein when combined with antibodies targeting CD33, CD123 and CSF1R leading to upregulation of T cell activation markers. Anti-P329G CAR T cells combined with PGLALA-mutated antibodies targeting CD33, CD123 und CSF1R achieved efficient in vitro cytotoxicity against tested AML cell lines and primary human AML blasts, while antigen-negative ALL cell line NALM-6 was not killed. Remarkably, effector functions of anti-P329G CAR T cells were similar to classical CAR T cells particularly when targeting CD33. This led to specific cytokine production when anti-P329G CAR T cells were co-cultured with respective antibodies and tumor cells. P329G-CAR T cells activated by recombinant CD33, CD123 or CSF1R showed no cytokine production in the presence of an antibody which was not recognizing the respective antigen. However, by switching the antibody after 24 h of stimulation towards the tumor antigen-targeting antibody, CAR T cell activation could be again achieved by the recombinant protein. Depleting the tumor antigen-targeting antibody after 24 h of stimulation decreased the cytokine production after 48 h and 72 h despite ongoing presence of the recombinant protein. This confirms the modularity and reversibility of this adaptor CAR T cell platform. Conclusions Taken together, anti-P329G CAR T cells combined with Fc-silenced tumor antigen-targeting IgG1 antibodies carrying the clinically validated PGLALA-mutations in the Fc part achieved profound effector functions against various human AML cell lines and primary AML blasts. The modular platform has the potential to overcome recent limitations of CAR T cell therapy in AML. These results support the rational for clinical translation of this novel and modular CAR T cell platform.

AML Inhibits Lipid Uptake By the Liver through an HGF/c-MET Dependent Mechanism Which Results in an Increased Supply of Free Fatty Acid to the Tumor

Acute myeloid leukemia (AML) requires a high metabolic turnover to allow for constant expansion. We and others have previously shown that primary human AML blasts rely on fatty acid (FA)-oxidation for survival and proliferation. (Shafat MS, et al. 2017, Jones CL, et al. 2018). The liver plays a major role in carbohydrate, protein, amino acid, and lipid metabolism (Hodson L, and Gunn PJ. 2019). Given that AML is a FA dependent tumor, we have studied the interactions between AML and the liver, and specifically how these affect lipid metabolism, the availability of pro-tumoral FFA and ultimately the mechanism by which this supports AML growth. To understand the role of lipid metabolism we used two syngeneic models of AML (HOXA9/Meis1 or MN1). When engrafted with AML cells, mice body weight during the latter stages of disease. After sacrifice, the size of epididymal and inguinal fat pads were significantly reduced in mice with AML compared to controls. Mice with AML also had elevated free fatty acid (FFA) and reduced glucose in the plasma. We and others have shown that AML induces lipolysis of white adipose tissue for uptake by blasts and resulting in tumor expansion (Shafat MS, et al. 2017, Ye H, et al. 2016). Since the liver is the master regulator of available FA in the plasma, we isolated livers from AML engrafted mice. Transcriptomics revealed down-regulation of genes involved in FA uptake and metabolism. RT-qPCR of RNA isolated from liver samples confirmed these results showing down-regulation of FA transport proteins, (FABP1, SLC27A2, SLC27A5, and CD36) and FA metabolism genes (CPT1, ACADM, and HMGCS2). Furthermore, primary hepatocytes isolated and cultured with AML conditioned media showed a down-regulation of FA uptake and metabolism genes. These data support the hypothesis that AML secretes a factor that alters lipid metabolism in the liver. To discover the factor that AML secretes, and which regulates FA uptake and metabolism in the liver, a proteome profile of the serum from AML engrafted mice was compared to conditioned media from AML cultured cells. Pathway analysis revealed hepatocyte growth factor (HGF), IL-1B and Granulocyte colony stimulating factor (G-CSF) as possible key factors involved in liver FA metabolism (Jing Y, et al. 2019, Kaibori M, et al. 2002). In vitro gene expressions analysis of primary hepatocytes revealed that addition of HGF to the medium, but not Il-1b or G-CSF, inhibited expression of FA uptake genes. In addition, using Seahorse mitochondrial stress kit we confirmed that primary hepatocytes decreased FA oxidation when cultured with AML conditioned media or HGF. Next, we confirmed that AML derived HGF was responsible for inhibition of hepatocyte FA genes by using a neutralizing antibody to HGF which blocked the transcriptional changes previously observed in hepatocytes cultured in AML conditioned media. This showed that AML derived HGF downregulates hepatocyte FA uptake and metabolism. Finally, to determine the role of AML in the uptake of FA by hepatocytes, cultures of primary murine hepatocytes were pretreated with AML conditioned media or HGF with and without neutralizing antibody. A BODIPY conjugated long chain FA was then added to the culture. Images analysis showed hepatocytes had reduced lipid uptake in the presence of AML condition media or HGF, but this was restored with the addition of HGF neutralizing antibody. Here we report that AML inhibition of lipid uptake in the liver, through an HGF dependent mechanism, increases the availability of plasma FA and forms a fundamental part of the AML disease. Ongoing work to develop our understanding of how AML increases access to FA to aid proliferation, may aid in the development of new therapeutics to target liver metabolism in combination with other AML therapies.

Bispecific CD33/CD123 targeted chimeric antigen receptor T cells for the treatment of acute myeloid leukemia

CD33 and CD123 are expressed on the surface of human acute myeloid leukemia blasts and other noncancerous tissues such as hemopoietic stem cells. On-target off-tumor toxicities may limit chimeric antigen receptor T cell therapies that target both CD33 and CD123. To overcome this limitation we developed bispecific human CD33/CD123 CAR T cells with an "AND" Logic gate. We produced novel CD33 and CD123 scFvs from monoclonal antibodies that bound CD33 and CD123 and activated T cells. Screening of CD33 and CD123 CAR T cells for cytotoxicity, cytokine production, and proliferation was performed and we selected scFvs for CD33/CD123 bispecific CARs. The bispecific CARs split 4-1BB costimulation on one scFv and CD3ζ on the other. In vitro testing of cytokine secretion and cytotoxicity resulted in selecting bispecific CAR 1 construct for in vivo analysis. The CD33/CD123 bispecific CAR T cells were able to control AML in a xenograft AML mouse model similar to monospecific CD33 and CD123 CAR T cells while showing no on-target off-tumor effects. Based on our findings, human CD33/CD123 bispecific CAR T cells are a promising cell-based approach to prevent AML and support clinical investigation.

Abstract 2591: Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models

Abstract Background: The Bcl-2 inhibitor venetoclax is approved in combination with hypomethylating agents such as azacitidine for the treatment of newly diagnosed elderly patients with AML; however, most patients eventually relapse, especially those with TP53 mutations who have poor prognosis. Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis. ZN-d5 and ZN-c3 are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. We show here that the combination is highly active in preclinical models of AML. Methods: Cell proliferation in tumor cell lines and AML patient’s samples were measured using CellTiter-Glo® (Promega). Relevant markers were measured by Western blotting. Anti-tumor efficacy was determined in cell line derived subcutaneous xenograft models of acute myeloid leukemia (AML) and in an AML patient-derived systemic xenograft (PDX) model. Flow cytometry was used for analysis of the PDX model to measure human AML blast populations. Results: The in vitro combination of ZN-d5 and ZN-c3 was tested in AML cell lines and showed synergistic or additive anti-proliferative activity which was independent of BCL-2 sensitivity in some cases. In addition, ZN-d5 in combination with ZN-c3 had a synergistic anti-tumor activity in vivo in cell line-derived AML models, such as MV4-11 and HL-60. Also, the triple combination of ZN-d5 + ZN-c3 + azacitidine in the HL-60 in vivo model resulted in synergistic anti-tumor efficacy and significant tumor growth inhibition compared to single- and double-agent treatments. The ZN-d5 + ZN-c3 combination was also tested in vitro in 29 AML patient-derived samples. Response to ZN-c3 alone was observed in 23/29 samples (IC50 < 450 nM), independent of TP53 mutation status. Response to ZN-d5 alone was observed in 17/29 samples (IC50 <200 nM). The combination resulted in synergy being observed in 12/29 models and had greater anti-proliferative activity than either single agent alone in all samples including those insensitive to Bcl-2 inhibition (IC50 > 10 μM). Combination of ZN-d5 and ZN-c3 was also highly active in an AML patient-derived PDX model, resulting in a 99.5% decrease in human CD45+ blast population in the bone marrow. Finally, the combination of ZN-d5 and ZN-c3 was active in 3/3 ex vivo experiments of patients derived AML samples who had progressed on venetoclax. Conclusions: These results justify testing the ZN-d5 and ZN-c3 combination or possibly the triple combination including azacitidine in AML patients independently of TP53 status. Due to synergism observed with these combination(s) and activity in samples from patients resistant to venetoclax, activity may be seen in patients with low sensitivity to Bcl-2 inhibitors or even in patients who progressed on venetoclax plus azacitidine. Citation Format: Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Joseph Pinchman, Brant C. Boren, Jianhui Ma, Petrus R. de Jong, Jiali Li, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2591.

Curcumin as an Epigenetic Therapeutic Agent in Myelodysplastic Syndromes (MDS).

Growth Factor Independence 1 (GFI1) is a transcription factor with an important role in the regulation of development of myeloid and lymphoid cell lineages and was implicated in the development of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Reduced expression of GFI1 or presence of the GFI1-36N (serine replaced with asparagine) variant leads to epigenetic changes in human and murine AML blasts and accelerated the development of leukaemia in a murine model of human MDS and AML. We and other groups previously showed that the GFI1-36N allele or reduced expression of GFI1 in human AML blasts is associated with an inferior prognosis. Using GFI1-36S, -36N -KD, NUP98-HOXD13-tg mice and curcumin (a natural histone acetyltransferase inhibitor (HATi)), we now demonstrate that expansion of GFI1-36N or –KD, NUP98-HODXD13 leukaemic cells can be delayed. Curcumin treatment significantly reduced AML progression in GFI1-36N or -KD mice and prolonged AML-free survival. Of note, curcumin treatment had no effect in GFI1-36S, NUP98-HODXD13 expressing mice. On a molecular level, curcumin treatment negatively affected open chromatin structure in the GFI1-36N or -KD haematopoietic cells but not GFI1-36S cells. Taken together, our study thus identified a therapeutic role for curcumin treatment in the treatment of AML patients (homo or heterozygous for GFI1-36N or reduced GFI1 expression) and possibly improved therapy outcome.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

Acute Myeloid Leukemia Cell-Intrinsic PD-1 Functions Promoting Leukemia Development By Recruiting SHP2

Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy. With current therapies, only approximately 30% of patients achieve long-term survival. Therefore, novel, more active and less toxic treatments are urgently needed. Programmed death-1 (PD-1) is a cell surface receptor that functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding of PD-1 to its ligand, programmed death-ligand 1 (PD-L1), activates downstream signaling pathways and inhibits T cell activation. Abnormally high PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape, and PD-1/PD-L1 immune checkpoint blockade has showed promising results in cancer patients. Recently, PD-1 expressed on melanoma cells was also shown to play a pivotal role in tumor growth. To date, in AML, the function of PD-1 has been mainly studied in the host T cells, while little is known regarding the role of PD-1 in AML cells. Herein, we examined the level and role of PD-1 in AML cells using AML murine model and patient samples.We used MLL PTD/WT/Flt3 ITD/ITD knock-in mouse in B6 background, a well characterized AML model, to study the expression and function of PD-1 in AML. Flow cytometric analysis of LSK (Lin -Sca-l1 +c-kit +) cells from the bone marrow (BM) of wild-type (WT, n=5) and AML (n=10) mice showed that 20.9%-61.9% of AML LSKs versus (vs) <5.0% of normal LSKs are PD-1 positive (P< 0.0001). Western blot and Q-RT-PCR analysis confirmed higher levels of PD-1 in AML LSKs than in normal LSKs. The PD-1 levels on LSKs increased over time and associated with disease progression. Similar results were obtained in AML patients, showing PD-1 + in 0.2%-14.6% of AML CD34 + cells vs < 1.0% of normal CD34 + cells (P< 0.05). Data analysis based on TCGA showed that higher PD-1 levels are associated with shorter survival in AML patients (P=0.0125).To assess the functional role of PD-1 in AML, we sorted PD-1 + and PD-1 - fractions fromAML LSKs and observed a lower frequency of quiescent cells (G0, 16.60% vs 44.87%, P< 0.05) and a higher cell growth rate in the PD-1 + vs PD-1 - cells. Further in vivo study showed that PD-1 + AML LSKs (CD45.2) generated higher white blood cell (WBC) counts (P< 0.0001), higher AML engraftment (P< 0.0001) and a shorter survival (median survival 57.5 vs >75 days, P< 0.001) in recipient mice (CD45.1) compared with PD-1 - AML LSKs. Similar results were observed in human samples. Compared to PD-1 - CD34 + cells, PD-1 + CD34 + cells are less quiescent and more proliferative (P< 0.01). PD-1 + AML blasts had higher engraftment rate (13.18% vs 2.68%, p=0.0002) and shorter survival (median survival: 27 vs 45 days, P= 0.0008) in NSGS mice than PD-1 - AML blasts.To evaluate if these in vivo differences observed in PD-1 + vs PD-1 - AML LSKs were mediated by interactions between PD-1 + AML and T cells, PD-1 + and PD-1 -LSKs from AML mouse were transplanted into T-, B- cell-deficient NSG mice. Recipient mice transplanted with PD-1 + AML LSKs had higher WBC counts (P< 0.01), higher AML engraftment (P< 0.0001) and a shorter survival (median survival: 76 vs >130 days, P< 0.0001) than recipients with PD-1 - AML LSKs, suggesting that these differences were T cell-independent.Next, we examined whether blocking PD-1 could affect leukemic cell growth. We sorted LSK cells from AML mice and performed colony forming cell (CFC) assay in the presence of anti-PD1 antibody or isotype antibody. Blocking PD-1 with anti-PD-1 antibody significantly suppressed CFC and cell growth in vitro but did not induce apoptosis compared to isotype control antibody. To explore the molecular mechanism by which PD-1 contributes to AML growth, we then sorted PD-1 + and PD-1 - LSKs from AML mice for molecular studies. Western blot assays revealed higher levels of SHP-2 and phosphorylated (p) -ERK in PD-1 + vs PD-1 - AML LSKs. We validated these results in primary human AML cells by immunofluorescence staining. Confocal microscopy of PD-1 + and PD-1 - human AML CD34 +cells demonstrated that PD-1 localized at the cell membrane and in the cytoplasm and p-ERK was markedly enhanced in the PD-1 + CD34 + cells.In conclusion, we showed here that a subpopulation of murine and human AML blasts expresses high levels of PD-1 which mediated disease initiation and growth through activation of the MAPK/ERK signaling pathways. PD-1 blocking antibody reversed these activities and might contribute to the clinical efficacy of anti-PD-1 therapy in AML. DisclosuresMarcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance.

Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.

SS30, a novel thioaptamer targeting CD123, inhibits the growth of acute myeloid leukemia cells

AimsCD123 represents an important acute myeloid leukemia (AML) therapeutic target. CD123 aptamers may potentially serve as tumor-homing ligands with excellent affinity and specificity for AML targeted therapy, but their complexity, laborious preparation and nuclease digestion limited pharmacological application. The aim of this study was to develop the first CD123 thioaptamer to overcome these obstacles. Main methodsFlow cytometry was utilized to assess the binding specificity, affinity and anti-nuclease ability of thioaptamer. CCK8, Annexin-V/DAPI, and colony forming assays were used to evaluate the anti-cancer ability of thioaptamer in vitro. The tumor volume, weights, survival rate, H&E staining of organs, and serum level of organ damage biomarkers of animal model were applied to investigate the anti-cancer ability of thioaptamer in vivo. Furthermore, we explored the binding mechanism between thioaptamer and CD123. Key findingsCD123 thioaptamer SS30 was able to bind to CD123 structure with high specificity in complex nuclease environment, the dissociation constant of 39.1 nM for CD123 peptide and 287.6 nM for CD123+ AML cells, while exhibiting minimal cross-reactivity to albumin. Furthermore, SS30 inhibited the proliferation and survival of AML cell lines and human AML blasts selectively in vitro (P < 0.01). In addition, SS30 prolonged the survival and inhibited tumor growth in a mouse xenograft tumor model in vivo. Of note, SS30 blocked the interaction between IL-3 and CD123, and decreased expression of p-STAT5 and p-AKT. SignificanceThe proliferation inhibition and nuclease resistance ability of SS30 made it as a more promising functional molecule for AML targeted therapy.

Abstract 3043: Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia

Abstract Myristoylation is required for biological activity of &amp;gt;200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML. Citation Format: John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia [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 3043.

Anthracycline-based consolidation may determine outcome of post-consolidation immunotherapy in AML

Consolidation chemotherapy in acute myeloid leukemia (AML) aims at eradicating residual leukemic cells and mostly comprises high-dose cytarabine with or without the addition of anthracyclines, including daunorubicin. Immunogenic cell death (ICD) may contribute to the efficacy of anthracyclines in solid cancer, but the impact of ICD in AML is only partly explored. We assessed aspects of ICD, as reflected by calreticulin expression, in primary human AML blasts and observed induction of surface calreticulin upon exposure to daunorubicin but not to cytarabine. We next assessed immune phenotypes in AML patients in complete remission (CR), following consolidation chemotherapy with or without anthracyclines. These patients subsequently received immunotherapy with histamine dihydrochloride (HDC) and IL-2. Patients who had received anthracyclines for consolidation showed enhanced frequencies of CD8+ TEM cells in blood along with improved survival. We propose that the choice of consolidation therapy prior to AML immunotherapy may determine clinical outcome.

Endogenous Tumor Suppressor microRNA-193b: Therapeutic and Prognostic Value in Acute Myeloid Leukemia.

Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio ± standard error, -0.56 ± 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms.

Abstract 3726: INCB52793 JAK1 inhibitor synergizes with ATRA to inhibit expansion of AML

Abstract Dysregulated JAK/STAT signaling is known to drive myeloproliferative neoplasms, and targeting JAK1 and JAK2 has led to improvement in morbidity and mortality in these diseases. While dose-dependent anemia and thrombocytopenia limit the use of JAK2 inhibition, selectively targeting JAK1 has been explored as a means to suppress inflammation and STAT-associated neoplastogenesis. Recently, INCB52793 was found to be 100-fold selective for JAK1 over JAK2, and it has recently been explored in the clinic in solid tumors and acute myeloid leukemia (AML). In a large high throughput screen, we detected synergistic effects between INCB52793 and all-trans retinoic acid (ATRA) in several non-promyelocytic AML cell lines. In another in vitro assay, human primary AML blasts exposed to INCB52793 exhibited a marked increase in both CD13 and CD86, two markers indicative of cellular differentiation. Given these findings, we tested this combination in an in vivo murine model of AML. Human leukemia cells were injected into the tail vein of sublethally irradiated NSGS mice which were then treated days 7-35 post-transplant with ATRA, INCB52793, ATRA/INCB52793, or vehicle. Weekly monitoring for peripheral human CD45+ cells revealed that the INCB52793/ATRA combination effectively decreased the expansion of leukemic cells. At 35-40 days, significant decreases in tumor burden were seen within the bone marrow (BM) and spleens of INCB62793/ATRA treated mice. Bone marrow and splenic cells were also analyzed by mass cytometry, simultaneously measuring 35 signaling, differentiation, and cell death attributes per-cell. The few remaining human cells in the INCB62793/ATRA combo group synergistically displayed 30-fold decreases in CD38, 8-fold increases in CD34, and attained high levels of p-STAT3 and p-STAT5, potentially implying a resistant progenitor population. Label free proteomics revealed significant fold changes in in vitro INCB52793/ATRA treated cells. Proteins related to cellular differentiation mechanisms, such as SMAD3, BCL11A, RUNX2, HNRNPLL, and SAMHD1, were elevated between 24 to 48 hours post treatment supporting our hypothesis that JAK1 inhibition enhanced ATRA induced differentiation. Targeting retinoic acid receptor and JAK1 together synergistically resulted in the decreased expansion of multiple AML cell lines, and preferential reduction of AML cells from the blood, spleen and bone marrow of treated mice in vivo. Common CD34-CD38+ tumor cells were eliminated, and rare remaining CD34+ AML cells displayed high p-STAT3 and p-STAT5 levels after INCB52793/ATRA therapy. While ATRA is a critical component in the therapy of acute promyelocytic leukemia (M3), it has not been successfully employed in other AML. These preliminary data represent a potential for INCB52793/ATRA therapy in non-M3 AML. Citation Format: Haley E. Ramsey, P. Brent Ferrell, Melissa A. Fischer, Agnieszka E. Gorska, Caroline Maier, Jeremy Norris, Melissa Farrow, Danielle Guiterrez, James Pino, Sandra Zinkel, Carlos Lopez, Holly Koblish, Matthew Stubbs, Peggy Scherle, Jonathan M. Irish, Richard Caprioli, Michael R. Savona. INCB52793 JAK1 inhibitor synergizes with ATRA to inhibit expansion of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3726. doi:10.1158/1538-7445.AM2017-3726

Endoglin (CD105) in AML: A Potential Novel Target for Therapeutic Intervention

Introduction: Successful treatment of acute myeloid leukemia (AML) remains a clinical challenge due to the toxicity of current therapies and the relatively poor responses to the current standard therapy. Thus, treatments that address novel therapeutic targets are urgently needed. Endoglin, also known as CD105, is a receptor of the transforming growth factor-beta (TGF-β) superfamily, found to be expressed in functional long-term repopulating hematopoietic stem cells. CD105 is expressed in several cancers, including AML. However, because CD105 expression has been studied mostly in the context of solid tumors and angiogenesis, relatively little is known about CD105 expression and its function in hematopoietic malignancies. Ongoing studies from our group have revealed distinct expression of this receptor in the majority of the blast population in cases of AML and acute lymphoid leukemia (ALL). Importantly, utilizing a xenograft model we found leukemia-forming-activity to be enriched in the CD105+ fraction of primary AML blasts. Based on these encouraging initial findings, we investigated the therapeutic potential of inhibiting CD105 signaling in AML using TRC105, a monoclonal antibody to CD105 that is currently being investigated in clinical trials for the treatment of multiple solid tumors.Materials and Methods: Sub-lethally irradiated NSG mice were intravenously injected with 5x105 human AML blast cells isolated from primary engrafted recipient mice. After infusion, mice were randomly divided into groups and treated with TRC105 (2mg/kg - IV. every 3 days) or IgG isotype control. Treatment started 2 or 30 days after the injection of AML blasts, and mice were monitored every 2 weeks for the presence of human CD45+ cells in the peripheral blood by FACS. Bone marrow and spleen were harvested at a predefined endpoint for analyses.Results: Inhibition of CD105 signaling using TRC105 suppressed the ability of AML blasts to give rise to leukemia in vivo. In mice that were treated on D+2 of AML inoculation, untreated or IgG control treated mice displayed a clear sub-population of human blast cells in the peripheral blood (~ 20%) at week 4, which increased significantly by week 8 (~60%) and 12 (~ 90%). In contrast, TRC105-treated mice exhibited significantly lower levels of human leukemia by week 4 (~5%), which remained low by week 12 (~10%). Similar results were observed in the spleen. Remarkably, treatment with TRC105 starting at day 30, once human engraftment was detected, inhibited further development of leukemia and increased survival. The sub-population of human blasts in peripheral blood in this group remained low by week 12 (~15%), unlike their untreated controls (~ 90%).Conclusion: Inhibition of CD105 using TRC105, a CD105 antibody in Phase 2 trials in solid cancer patients, inhibited the ability of injected human primary AML blasts to produce leukemia in NSG mice. Therefore TRC105 represents a potential novel therapy for AML. This is of particular relevance if we consider the population of AML patients that are either unable to receive intensive chemotherapy (i.e., elderly AML) or those who do not respond to standard chemotherapy. DisclosuresTheuer:TRACON Pharmaceuticals: Employment. Perlingeiro:TRACON Pharmaceuticals: Research Funding.

EGFL7 Antagonizes NOTCH Signaling, Stimulates Blast Proliferation and Confers Poor Prognosis in Cytogenetically-Normal Acute Myeloid Leukemia (CN-AML)

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein and plays an important role in angiogenesis by regulating the growth, proliferation and migration of endothelial cells. Recent studies in solid tumors have shown that EGFL7 is overexpressed and is associated with a more aggressive disease phenotype. Whether EGFL7 plays a similar role in blood cancers such as acute myeloid leukemia (AML) however, has not been previously reported. To investigate the association of EGFL7 expression with outcome in AML, we measured EGFL7 mRNA expression in newly diagnosed older (≥60 years, n=126) CN-AML patients. In these patients, those with high EGFL7 expression were less likely to achieve CR (52% v 76%, P=.009). Patients with high EGFL7 expression status had shorter event-free survival (5-year rates: 6% v 13%, P=.03) and overall survival (5-year rates: 10% v 16%, P=.009) than patients with low EGFL7 expression status.To validate our clinical data we measured EGFL7 mRNA in primary AML blasts (n=11) compared to normal bone marrow (NBM) (n=5) using RT-PCR, and found a ~2.4 fold-increase in the AML samples, P<0.05. These results were consistent with the protein levels, measured using western blots. Using both ELISA and western blotting, we observed significant increased levels of EGFL7 protein in the medium of cultured primary AML cells (n=3, 31-443 pg/mL) compared to media alone. Importantly, detection of serum EGFL7 levels by ELISA revealed variable but increased levels of EGFL7 in the sera from 3 of 5 (P<0.05) AML patients tested compared with sera from normal healthy controls (n=7). To investigate the functional consequences of EGFL7 stimulation on blast growth we treated primary AML blast cells from three patients with recombinant EGFL7 (rEGFL7) protein and performed colony forming unit (CFU) assays. Treatment with rEGFL7 led to an increase in the number of CFUs (P<0.05) and was associated with increased phosphorylation of AKT. In addition, under starvation conditions, recombinant EGFL7 protein alone promoted the survival and proliferation of MLL-PTD AML cells (n=3) but not normal BM cells (n=3) (P<0.05). To explore the underlying molecular mechanism(s) by which EGFL7 maintains the survival and proliferation of AML cells, we screened for proteins that interact with EGFL7 by using a protein antibody array on human primary AML blasts. This screen revealed that EGFL7 binds to multiple components of key signal transduction pathways important for leukemia, including NOTCH. Previous studies by Lobry C et al., (JExpMed 2013) demonstrated that although NOTCH2 and to a lesser extent, NOTCH1, mRNA was relatively abundant, NOTCH signaling was inhibited in AML blasts. Re-activation of the canonical NOTCH pathway, released a block in differentiation resulting in eradication of the leukemic blasts. Schmidt MH et al., (Nat Cell Biol. 2009) demonstrated that EGFL7 was capable of binding NOTCH receptors and blocking their subsequent activation. Therefore, we hypothesized that one mechanism by which the NOTCH pathway is kept inactive in AML is through autocrine binding of EGFL7 protein to NOTCH receptors. Co-immunoprecipitation assays confirmed binding of EGFL7 to NOTCH2 in both human primary AML cells (n=3, P<0.05) and mouse MLL-PTD AML cells (n=3, P<0.05). Moreover, stimulation of AML blasts with rEGFL7 protein suppressed the cleavage and subsequent activation of NOTCH2, resulting in decreased transcription of NOTCH downstream target genes, HES1 and NRARP (n=3, P<0.05). In summary, we show here that high EGFL7 is associated with worse prognosis in patients with CN-AML, and that EGFL7 is expressed and secreted by the AML cells in an autocrine fashion, promoting their growth in part through antagonizing NOTCH2 activation. Targeting EGFL7 with antibodies to reduce NOTCH binding might represent a novel therapeutic approach to reactivate NOTCH signaling, allowing for blast differentiation and elimination of AML. DisclosuresNo relevant conflicts of interest to declare.

The High Affinity CXCR4 Inhibitor, BL-8040, Induces Apoptosis of AML Blasts and Their Terminal Differentiation By Blocking AKT/ERK Survival Signals and Downregulating BCL-2, MCL-1 and Cyclin-D1 through Regulation of Mir-15a/16-1 Expression

Background: Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation and survival of hematopoietic stem and progenitor cells. The chemokine CXCL12 and its receptor CXCR4 are key players in the survival, bone marrow (BM) retention and the maintenance of AML blasts in their stemness state. CXCR4 overexpression is associated with poor prognosis in AML patients. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy.Objective: To study the effect of the CXCR4-antagonist, BL-8040, on the survival of human AML blasts and to investigate the molecular mechanism by which inhibition of CXCR4 signaling leads to leukemia cell death.Methods: Human AML cell lines and human primary AML samples were used for in vitro studies. The in-vivo effect of BL-8040 was tested using the MV4-11, U-937, THP-1 cells and human primary AML cells engrafted in NOD scid gamma (NSG) mice.Results: We found that BL-8040 directly induced apoptosis of AML cells both in FLT3-ITD and FLT3-WT AML, in-vitro and in-vivo. BL-8040 treatment triggered mobilization of AML blasts from their protective BM microenvironment and induced their terminal differentiation, in-vitro and in-vivo. The apoptosis of AML cells induced by BL-8040 was attributed to miR-15a/miR-16-1 up-regulation resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin-D1. The increase in miR-15a/miR-16-1 levels directly induced AML cell death. Moreover, CXCR4 blockade by BL-8040 also inhibited survival signals by the ERK/AKT kinases enhancing the apoptosis effect. Survival of AML cells was found to be dependent on BCL-2 as demonstrated by the ability of the BCL-2 inhibitor, ABT-199, to induce dose dependent apoptosis in vitro. It was reported that the MCL-1 protein plays a key role in acquiring resistance to ABT-199. We found that BL-8040 synergizes with ABT-199 in inducing AML cell death. This could be attributed to the reduction of both, AKT/ERK and MCL-1 levels, by treatment with BL-8040. In addition, BL-8040 synergizes with the FLT3 inhibitor AC220 in the induction of AML cell death both in-vivo and in-vitro. The combined treatment of BL-8040 and AC220 was found to prolong survival and reduce minimal residual disease in-vivo. Interestingly, the combined treatment was also associated with a significant reduction in the expression of BCL-2 and ERK signaling.Conclusions: BL-8040 can be a potential therapeutic option in AML by targeting not only AML anchorage in the BM but also AML survival and differentiation. Our results demonstrate that BL-8040 in AML regulates the expression of miR-15a/16-1 and their target genes BCL-2, MCL-1 and cyclin-D1. Furthermore, these results indicate that the CXCR4 antagonist, BL-8040 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 pathway and inhibition of the ERK/AKT survival signaling cascade in AML cells. Our results provide rational for combination of BL-8040 with ABT-199 to overcome potential acquired resistance to ABT-199 in AML patients. The synergistic effect of BL-8040 with AC220 could provide a rational basis for the combination of BL-8040 with FLT3 inhibitors in FLT3-ITD AML patient population. [Display omitted] [Display omitted] [Display omitted] DisclosuresAbraham:Biokine Therapeutics Ltd: Employment. Bulvik:Biokine Therapeutics Ltd: Employment. Wald:Biokine Therapeutics Ltd: Employment. Eizenberg:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Peled:Biokine Therapeutics Ltd: Consultancy, Employment.

8-Chloro-Adenosine Inhibits Molecular Poor-Risk Acute Myeloid Leukemia (AML) and Leukemic Stem Cells (LSC) Growth and Synergizes with the BCL-2 Inhibitor Venetoclax (ABT-199)

Only a minority of AML patients are cured with currently available chemotherapy regimens. Thus, new drugs with novel mechanisms of action are urgently needed. In previous proof-of-concept studies, the halogenated ATP analog 8-chloro-adenosine (8-Cl-Ado) has shown preclinical activity against a variety of solid tumors and hematologic malignancies, favorable pharmacokinetic and pharmacodynamic profiles and feasibility in a first-in-man phase I clinical trial in chronic lymphocytic leukemia. However, little is known regarding the activity of this compound in AML. In contrast to other nucleoside analogs used for treatment of AML, 8-Cl-Ado is metabolized by adenosine kinase and its triphosphate derivative is incorporated into RNA and significantly inhibited RNA synthesis in AML cells (KG-1a and MV4-11 cell lines and primary bone marrow samples) in a dose-dependent manner (300 nM to 1 uM) after 24 h exposure as compared to vehicle-treated controls (p<0.05). Similarly, 20-80% inhibition of total RNA synthesis was observed in AML patient blasts, regardless of the harbored gene mutations or cytogenetic aberrations. In contrast, 24 h exposure to 8-Cl-Ado did not significantly inhibit DNA synthesis compared to vehicle at all concentrations tested (p>0.25 at all concentrations tested). 8-Cl-Ado was metabolized into its cytotoxic triphosphate, 8-Cl-ATP, which accumulated to intracellular concentrations of more than 600 uM after 12 h exposure to 10 uM 8-Cl-Ado. Accumulation of 8-Cl-ATP was associated with >20% reduction of endogenous ATP compared to control-treated cells (p<0.05). In regard to antileukemia activity, 8-Cl-Ado inhibited growth of several AML cell lines (MOLM-13, MOLM-14, KG-1a, MV4-11, OCI-AML3) with IC50s ranging from 0.2 uM to 1.4 uM after 72 h of treatment and that of primary AML blasts including those harboring the poor-risk FLT3-ITD mutation (IC50: 800 nM for FLT3-ITD-positive blasts). In an orthotopic mouse model where FLT3-ITD-positive MOLM-14 AML cells were xenografted, animals treated with 50 mg/kg/day through an implanted osmotic pump had >70% reduction in tumor mass compared to vehicle-treated controls after 16 days of treatment (p<0.05). Importantly, 24 h pre-treatment of human LSC-enriched CD34+CD38- blasts with 5 uM 8-Cl-Ado resulted in a significant inhibition of their colony forming ability compared to vehicle-treated controls (>50% reduction of colonies after 14 days, p<0.05). To study the effect of 8-Cl-Ado on primary AML in vivo, sub-lethally irradiated Rag-2/gamma(c) double-knockout immunodeficient mice were injected retro-orbital with ten million primary human AML blast cells pre-treated with 5 uM 8-Cl-Ado or vehicle control 24 h prior to i.v. injection. Significant longer survival was observed of mice engrafted with drug-treated cells compared to those engrafted with vehicle-treated controls (p<0.0004). Given the previously reported higher expression of the antiapoptotic BCL-2 protein and the implication that this had on treatment resistance in AML, combination strategy using 8-Cl-Ado together with the BCL-2 inhibitor venetoclax was investigated and showed strong synergistic, anti-proliferative effects in both AML cell lines and primary AML blasts carrying a variety of mutations and cytogenetic aberrations. Combination index value calculation and isobologram display demonstrated strong synergy between venetoclax and 8-Cl-Ado in all 4 AML cell lines and 2 primary AML samples investigated, at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90). We also show that, in KG-1a and MV4-11 cells, 8-Cl-Ado and venetoclax synergize in the inhibition of intracellular ATP pools. AML cell lines were treated for 6h with 10 uM 8-Cl-Ado, 250 nM venetoclax or both, before measurement of intracellular ATP. While single agent treatment decreased intracellular ATP levels in both cell lines by about 20% (8-Cl-Ado) to 40% (venetoclax), combination treatment lowered ATP levels by about 70% (KG-1a) to 90% (MV4-11). Taken together, 8-Cl-Ado is a promising agent with a unique RNA and ATP-targeting mechanism of action, excellent toxicity profile and encouraging preclinical anti-leukemia activity in AML and synergizes with the BCL-2 inhibitor venetoclax in vitro. Based on these results, we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory AML and planned further combination with BCL-2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Leukemia Stem Cells Are Characterized By CLEC12A Expression and Chemotherapy Refractoriness That Can be Overcome By Targeting with Chimeric Antigen Receptor T Cells

Chemo-refractory acute myeloid leukemia (AML) is associated with poor prognosis and treatment options are extremely limited. Most of these patients are ineligible for allogeneic stem cell transplantation. Chemo-refractory AML is thought to arise due to selection pressure of resistant clones from prior use of chemotherapy or in some cases pre-exist due to properties of the leukemic stem cells (LSC). CLEC12A (also known as CLL1) has previously been described as being selectively over expressed in LSCs. Successful modalities to target CLEC12A and eradicate the LSC would overcome chemo-refractoriness in AML and would represent a vertical advance in the field.In this study, we confirm that CLEC12A is heterogenously expressed on AML blasts and over-expressed on AML LSC. We also show that CLEC12A is overexpressed on bone marrows from patients with AML that fail to achieve a complete remission after induction chemotherapy, suggesting that it could be a marker for residual disease that is refractory to chemotherapy. We then separated AML blasts into CLEC12A positive or negative cells by magnetic sorting. CLEC12A positive blasts selected from AML patients were more resistant to chemotherapy compared to CLEC12A negative blasts (20% killing of CLEC12A positive AML cells versus 43% of CLEC12A negative AML cells when cultured with cytarabine 10 µg/ml, P=0.01). This finding was confirmed by using the AML MOLM14 cell line engineered to overexpress CLEC12A. CLEC12Ahigh MOLM14 cells were more resistant to chemotherapy compared to wild type MOLM14 cells (P=0.003). We then evaluated CLEC12A resistance to chemotherapy in a patient derived AML xenograft model. We found a relative increase in CLEC12A positive cells post Ara-C induction chemotherapy in AML xenograft models (Figure 1). The observation that CLEC12A positive cells are more resistant to chemotherapy provided a solid rationale to target CLEC12A with chimeric antigen receptor T (CART) cells. We therefore developed a second generation CLEC12A directed CAR construct using CD3z and 41BB costimulatory domains and generated CLEC12A CART cells by lentiviral transduction with this construct. Upon incubation with primary AML samples or AML cell lines, CLEC12A CART cells resulted in modest effector functions, due to the heterogeneity of CLEC12A expression on AML blasts. However when CLEC12A overexpressed MOLM14 cell line or CLEC12Apos selected leukemic cells were used as targets, CLEC12A-CART cells resulted in potent cytotoxicity, proliferation and cytokine production, indicating that CLEC12A-CART cells are more specific for LSC.To test the in vivo anti-leukemic activity of CLEC12A CARTs, we used primary human AML blasts xenografted into NSG-S mice (NOD-SCID-γc-/-, additionally transgenic for human stem cell factor, IL3 and GM-CSF). Treatment with CLEC12A CART (single dose, 1x105 total T cells via tail vein injection) resulted in modest activity against AML when employed as monotherapy. To investigate the potential role of CLEC12A CART cells in eradication of MRD and LSC, mice were treated first with chemotherapy (cytarabine 60 mg/kg intraperitoneal injection daily for 5 days) followed by a single dose (1x105 total T cells via tail vein injection) of either CLEC12A CARTs or control untransduced T cells (UTD). Treatment with CLEC12A CART cells resulted in eradication of leukemia and prolonged survival in these mice (overall survival at 200 days of 100% after CLEC12A CARTs compared to 20% after UTD, p=0.01, Figure 2).In conclusion, our preclinical studies reveal that CLEC12A positive cells in leukemia are resistant to chemotherapy and can be successfully targeted with CART cells. CLEC12A CART cells can potentially be employed as a consolidation regimen after induction chemotherapy to eradicate LSC and MRD in AML. [Display omitted] [Display omitted] DisclosuresKenderian:Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Singh:Novartis: Employment. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Johnson & Johnson: Research Funding; Pfizer: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.