Killing Of Acute Myeloid Leukemia Cells Research Articles

Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using cross-over dual-variable domain (CODV) format for acute myeloid leukemia (AML) treatment

ABSTRACT Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.

Induced CD25+CD127dim Γδ Tregs in Acute Myeloid Leukemia Suppress the Activity of Normal Αβ T Cells

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy in adults with dismal outcomes. γδ T cells have been reported to exert effective anti-tumor activities on various types of solid tumors and hematologic malignancies, and thus become a potential weapon used in the treatment of cancer. However, compared to the striking results demonstrated in vitro and in mouse models, the clinical efficacy of γδ T cells-based immunotherapy is disappointing. Meanwhile, recent studies identified a pro-tumor role of γδ T cells in some types of solid tumors. Whether γδ T cells could be transformed from warriors to foe in the environment of leukemia is unclear. In the current study, we aim to dissect the abnormal changes and functions of intrinsic γδ T cells in the context of AML. Considering bone marrows (BM) are the primary tumor foci of AML and likely provide more representative immune characteristics during leukemia development, the immunophenotyping analyses of γδ T cells in BM from newly diagnosed AML patients (n = 21) were detected using flow cytometry, compared with healthy individual controls (n = 21). We observed that the proportion of γδ T cells and the composition of Vδ1+ and Vδ2+ subgroups were comparable between AML patients and healthy controls. Interestingly, a dramatically elevated subpopulation in γδ T cells expressing CD25 and dim CD127 was observed in AML patients compared with that in healthy controls (3.2% versus 0.8%, P < 0.001). Since their phenotypes are akin to the conventional CD4+ regulatory T cells (Tregs), these CD25+CD127dim γδ T cells are called γδ Tregs here. Paired comparison was also performed on AML patients at diagnosis (n = 13) and completed remission (CR) after chemotherapy (n = 13). A significant decrease in proportion of γδ Tregs was observed at CR, compared with that at newly diagnoses (3.1% versus 0.4% (P < 0.001). These results suggest that this aberrant γδ T subset, γδ Tregs, is induced in the microenvironment of AML and can be reversed after effective therapy for elimination of leukemia blasts. To further explore the direct effect of AML blasts on γδ T cells, primary malignant cells were sorted from AML patients, and co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. As expected, γδ Tregs was significantly induced and the frequency was continuously elevated from 0.2% to 4.0% following the increased ratios of AML cells (P < 0.001). These results confirmed that AML cells were capable to trigger the transformation of normal γδ T cells into CD25+CD127dim γδ Tregs. To determine the function of this special γδ T subset, CD25+CD127dim γδ Tregs were expanded in vitro and co-cultured with AML cell lines, U937 cells. Unlike pamidronate-expanded cytotoxic Vδ2+ T cells, γδ Tregs lost the ability of killing AML cells. Furthermore, we explored the impact of γδ Tregs on the anti-AML activity of normal αβ T cells. γδ T cell-depleted PBMCs (more than 90% are αβ T cells) of healthy donors were co-cultured with U937 cells, with or without autologous γδ Tregs. Flow cytometry analysis showed that 7-AAD-positive fraction in U937 cells was dramatically increased from 0.3% to 7.6% after co-culture with PBMCs and without γδ Tregs. Whereas the mortality of U937 cells was gradually declined (from 7.6% to 4%) in the presence of γδ Tregs at increasing ratios (P = 0.031). During this process, γδ Tregs remarkably downregulated the expressions of HLA-DR and CD38, and productions of TNF-α and IFN-γ in both CD4+ and CD8+ αβ T cells. These findings indicated that γδ Tregs significantly impaired the cytotoxic ability of effector αβ T cells on AML cells, suggesting γδ Tregs play a suppressive role in the anti-leukemia immunity. In summary, we first reported the significant induction of CD25+CD127dim γδ Tregs in the primary BM of AML patients. These γδ Tregs did not exert anti-leukemia activity and attenuated the cytotoxic effect of normal αβ T cells. Our findings will help advance the current understanding of immune mechanism associated with leukemia cell evasion. It also proposes that boosting the cytotoxic γδ T cells should be coupled with inhibition of the suppressive γδ Tregs, for future improvement of γδ T-cell-based immunotherapy. Disclosures No relevant conflicts of interest to declare.

A Phase 1a/b Dose Escalation Study of the MYC Repressor Apto-253 in Patients with Relapsed or Refractory AML or High-Risk MDS

A Phase 1a/b Dose Escalation Study of the MYC Repressor Apto-253 in Patients with Relapsed or Refractory AML or High-Risk MDS

A trispecific killer engager molecule against CLEC12A effectively induces NK-cell mediated killing of AML cells

The low five-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using Natural Killer (NK) cells, we have developed a trispecific killer engager (TriKE™) molecule containing a humanized anti-CD16 heavy chain camelid single domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK cell specific proliferation, enhanced NK cell activation and killing of both AML cell lines and primary patient derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in pre-clinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.

Dendrogenin A Enhances Anti-Leukemic Effect of Anthracycline in Acute Myeloid Leukemia.

Simple SummaryRecently, several molecules have improved the clinical outcome of acute myeloid leukemia (AML) patients. Despite these recent advances, their prognosis remains poor and new strategies to improve the standard anthracycline and Ara-C-based chemotherapy are needed. We recently published that dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor-suppressor properties, can potentiate the effect of Ara-C to kill AML cells. In this study, we find that DDA can also potentiate anthracycline against AML. The potentiation of Ara-C by DDA is due to a switch from a protective autophagy to a deadly autophagy. Regarding anthracyclines, the potentiation of daunorubicin is caused by the modulation of the efflux by the PgP pump, and that of idarubicin, to an increase in DNA damage and to the induction of a rapid and lethal autophagy. This is caused by rapid modulation of AKT/mTOR and JNK activity, two major pathways involved both in DNA repair and lethal autophagy.Dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor suppressor properties, has recently been shown to exhibit strong anti-leukemic activity in acute myeloid leukemia (AML) cells by triggering lethal autophagy. Here, we demonstrated that DDA synergistically enhanced the toxicity of anthracyclines in AML cells but not in normal hematopoietic cells. Combination index of DDA treatment with either daunorubicin or idarubicin indicated a strong synergism in KG1a, KG1 and MV4-11 cell lines. This was confirmed in vivo using immunodeficient mice engrafted with MOLM-14 cells as well as in a panel of 20 genetically diverse AML patient samples. This effect was dependent on Liver X Receptor β, a major target of DDA. Furthermore, DDA plus idarubicin strongly increased p53BP1 expression and the number of DNA strand breaks in alkaline comet assays as compared to idarubicin alone, whereas DDA alone was non-genotoxic. Mechanistically, DDA induced JNK phosphorylation and the inhibition of AKT phosphorylation, thereby maximizing DNA damage induced by idarubicin and decreasing DNA repair. This activated autophagic cell death machinery in AML cells. Overall, this study shows that the combination of DDA and idarubicin is highly promising and supports clinical trials of dendrogenin A in AML patients.

Abstract A30: Targeting of the mTOR pathway in human acute myeloid leukemia cells using functionalized gold nanoparticles

Abstract Specific targeting of fundamental biochemical/signaling pathways in human malignant cells with the purpose of recognition and delivery of cytotoxic drugs into them is a very promising but not well-developed strategic approach. It is a major focus of current molecular cancer research, nanomedicine, biotechnology, and synthetic biology. mTOR pathway is a master regulator of metabolism and cell growth in the majority of malignant cells. Especially this applies to cancerous myeloid cells where it determines the ability of the cells to survive and evade the host immune surveillance. Therefore, specific targeting of this pathway could be a novel strategy for treatment of acute myeloid leukemia (AML), a blood/bone marrow cancer that can rapidly progress into a systemic and often fatal malignancy. In this study we demonstrated for the first time a fundamentally novel approach for specific targeting of human AML cells by functionalized gold nanoparticles. The immune receptor Tim-3 is highly expressed in human AML cells. It is one of the key members of the Tim-3/galectin-9 autocrine pathway that plays a pivotal role in AML cell survival and immune evasion (1). Rapamycin inhibits the mTOR activity, thus affecting translational pathways in AML cells. Inhibition of the mTOR leads to a rapid killing of AML cells. Using these nanomaterials, we managed to successfully deliver rapamycin into AML cells, reaching attenuation of the mTOR activity. Concentration of rapamycin required to reach such a strong effect was at least 50 times lower compared to that of free rapamycin required to achieve the similar result. We therefore concluded that our technology is of potential use for specific targeting of AML cells for the purpose of diagnosis and possibly therapy (2). The nanoconjugates developed in our work can be used to specifically identify malignant blood cells, thus allowing rapid and robust AML diagnosis. Citation Format: Inna M. Yasinska, Bernhard F. Gibbs, Luca Varani, Rohanah Hussain, Giuliano Siligardi, Elizaveta Fasler-Kan, Luigi Calzolai, Vadim V. Sumbayev. Targeting of the mTOR pathway in human acute myeloid leukemia cells using functionalized gold nanoparticles [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A30.

Abstract 2048: Discovery of induction and release of IL-1b are unique and on-target effects of GSPT1 degradation that provide potential mitigation strategies to hypotension in the CC-90009-AML-001 phase 1 trial

Abstract CC-90009 is a novel cereblon (CRBN) E3 ligase modulator (CELMoD) that has demonstrated antileukemic activity and is under investigation in the CC-90009-AML-001 phase I trial (NCT02848001) in patients with relapsed or refractory acute myeloid leukemia (AML). In preclinical studies, CC-90009 drives the binding of translation termination factor G1 to S phase transition I (GSPT1) to CRBN. This results in ubiquitination and proteasome-dependent degradation of GSPT1, leading to activation of the integrated stress response, inhibition of nonsense-mediated decay, and induction of apoptosis. Hypotension, contemporaneous with blast and white blood cell (WBC) decreases, has emerged as a dose-limiting toxicity (DLT) in this ongoing phase I trial. Based on clinical observations, CC-90009-induced cytokine release during leukemic cell death was investigated as a potential cause of hypotension. Using electrochemiluminescence, flow cytometry and western blots, CC-90009-induced IL-1b release was observed in AML cell lines and primary AML bone marrow mononuclear cells. Induction of IL-1b was typically observed after caspase 3 and 8 activation, suggesting a cell-death-related mechanism consistent with clinical findings of hypotension reported after rapid WBC loss. In vitro models showed compounds leading to GSPT1 degradation were more potent IL-1b inducers compared to compounds with other mechanisms, including standard-of-care agents for AML, other cytotoxic agents, protein translation inhibitors, and unfolded protein response inducers. IL-1b induction as a downstream result of GSPT1 degradation was further confirmed by cells expressing non-degradable GSPT1 mutant protein where compound treatment did not induce IL-1b. CC-90009 activates the GCN2 pathway, resulting in caspase-3 and -8-mediated apoptosis. Caspase 8 is known to directly process pro-IL-1b into mature IL-1b. Using an isogenic MV4-11 Cas9 cell pair with wild-type vs null GCN2 status, GCN2 activation downstream of GSPT1 degradation was shown to be required for pro-IL-1b upregulation and caspase 8 activation. Furthermore, genetic and pharmacologic suppression of caspase 8 revealed that its activity was necessary, but not sufficient, for IL-1b release by CC-90009. Of note, dexamethasone (DEX) dampened CC-90009-mediated IL-1b induction without altering the rate or depth of AML cell killing, providing a mechanism-based strategy to manage CC-90009-induced hypotension. The ongoing trial was amended to allow the use of prophylactic DEX; dose escalation is still ongoing, including dose levels higher than the non-tolerated dose defined prior to mandating the use of prophylactic DEX. The insights derived from this study have facilitated GSPT1 targeting in AML therapy by enabling the testing of higher drug exposures. Citation Format: Tsun-Wen Sheena Yao, Yumin Dai, Carla Guarinos, Manuel Sanchez, Alicia Benitez, Hongbin Wang, Soraya Carrancio, Tonia Buchholz, Gang Lu, Michael Pourdehnad, Daniel Pierce, Jinhong Fan. Discovery of induction and release of IL-1b are unique and on-target effects of GSPT1 degradation that provide potential mitigation strategies to hypotension in the CC-90009-AML-001 phase 1 trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2048.

5-Azacitidine Induces NOXA to Prime AML Cells for Venetoclax-Mediated Apoptosis.

Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown. We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity. In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza-induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be "priming" the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy. These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.

Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway.

Acute myeloid leukemia (AML) is a common type of hematological malignancy that can progress rapidly. AML has a poor prognosis and a high incidence of relapse due to therapeutic resistance. Azelaic acid (AZA), a small molecular compound is known to exhibit antitumor effect on various tumor cells. This study aimed to evaluate the antiproliferative and immunoregulatory effects of AZA against AMLviathe activation of the notch signaling pathway. We found that AZA can inhibit the proliferation of AML cells. In addition, laser confocal microscopy showed AZA-treated AML cells began to swelling and undergo cytoplasmic vacuolization. Importantly, AZA promoted the proliferation of NK and T cells and increased the secretion of TNF-αand IFN-γ. AZA also increased the expression levels of CD107a and TRAIL in NK cells, and CD25 and CD69 in T cells to influence their activation and cytotoxic ability. AZA-treated NK cells can kill AML cells more efficiently at the single-cell level as observed under the microfluidic chips. Further mechanistic analysis using protein mass spectrometry analysis and Notch signaling reporter assay demonstrated that Notch1and Notch2 were up-regulated and the Notch signaling pathway was activated. Moreover, combining AZA with the Notch inhibitor, RO4929097, decreased the expression of Notch1and Notch2, and downstream HES1 and HEY1, which rendered AML cells insensitive to AZA-induced apoptosis and alleviated AZA-mediated cytotoxicity in AML. In vivo, AZA relieved the leukemic spleen infiltration and extended the survival. The percentage of CD3-CD56+NK cells and CD4+CD8+T cells as well as the secretion of cytotoxic cytokines was increased after the treatment of AZA. The overall findings reveal that AZA is a potential Notch agonist against AML in activating the Notch signaling pathway.

Targeting Intracellular WT1 in AML Utilizing a T Cell Bispecific Antibody Construct: Augmenting Efficacy through Combination with Lenalidomide

Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Xu:Roche: Employment, Equity Ownership, Patents & Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents & Royalties. Sam:Roche: Employment, Equity Ownership, Patents & Royalties. Pulko:Roche: Employment, Equity Ownership, Patents & Royalties. Schönle:Roche: Employment, Equity Ownership, Patents & Royalties. Challier:Roche: Employment, Equity Ownership, Patents & Royalties. Carpy:Roche: Employment, Equity Ownership, Patents & Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.

The Splicing Modulator GEX1A Exhibits Potent Anti-Leukemic Activity Both in Vitro and In Vivo through Inducing an MCL1 Splice-Switch in Pre-Clinical Models of Acute Myeloid Leukemia

Background: Overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl1 is commonly detected in acute myeloid leukemia (AML) and is correlated with poor patient prognosis. Fortunately, in most AML samples, the expression of pro-apoptotic proteins, including Bax, Bak and Bim, is also increased. This is likely due to a feedback mechanism, which results in AML cells that are particularly vulnerable to treatment with targeted inhibition of anti-apoptotic proteins. However, challenges arise when the important pro-survival proteins named above are either poorly targeted by specific inhibitors (such as BH3-mimetics), as in the case of Mcl1, or when the use of these inhibitors results in severe thrombocytopenia, as in the case of Bcl-xL. Therefore, the development of novel medications to target such molecules with high specificity and low toxicity is of crucial importance. GEX1A, a splicing modulator, induces a large shift in the pattern of exon skipping and intron retention events by inhibiting the SF3B1-PHF5A complex of the U2 snRNP. While GEX1A has shown pre-clinical efficacy in some carcinoma cell lines containing various spiceosomal mutations, the in vitro and in vivo effects of GEX1A in leukemic cells which lack spiceosomal mutations are largely unknown. Experimental procedures: The IC50 of 18 established AML cell lines was determined by treating the cells with GEX1A in vitro using a series of 10-fold dilutions; cell viability was then examined using quantitative colorimetric assays. The in vivo anti-leukemic efficacy of GEX1A was evaluated using xenograft models generated in NSG mice injected with the GEX1A-sensitive human AML cell lines KOPN8, Molm-13, and MV4:11. The molecular mechanism by which GEX1A kills AML cells was then studied. Results: Based on their IC50, 18 established leukemic cell lines can be divided into high (LD50 < 100 nM), moderate (LD50 = 100-500 nM), and low sensitivity (LD50 > 500 nM) groups. We next determined the tolerable dose of GEX1A for in vivo mouse treatment. Intraperitoneal injection of GEX1A significantly improved the survival of leukemic mice compared to the vehicle control group (see Figure). Mechanistically, we found that GEX1A kills leukemic cells by inducing the production of pro-apoptotic Mcl1-S protein at the expense of the pro-survival Mcl1-L protein due to the alternative splicing of the Mcl1 gene. In addition, we found that high levels of Bcl-xL protein predict resistance of leukemic cells to GEX1A treatment. Furthermore, synergistic activity was observed in 2/6 cell lines treated with GEX1A and ABT-263, an inhibitor of Bcl-2/Bcl-xL/Bcl-w, and 6/6 cell lines treated with GEX1A and A-1155463, a highly potent Bcl-xL inhibitor. Conclusion: GEX1A is a novel splicing modulator that shows potent anti-leukemic activity. GEX1A kills leukemic cells by inducing a splicing isoform switch of the Mcl1 gene to produce a pro-apoptotic Mcl1-S protein at the expense of the pro-survival Mcl1-L protein in such cells. GEX1A and a Bcl-xL-specific inhibitor were also combined to show synergistic effects on leukemic cells. Our results indicate that GEX1A may be an effective treatment for leukemic patients when combined with a specific Bcl-xL inhibitor. We are actively evaluating our combination treatment using primary patient samples in in vivo xenograft models. Disclosures No relevant conflicts of interest to declare.

A Phase 1a/b Dose Escalation Study of Apto-253 in Patients with Relapsed or Refractory AML or High-Risk MDS

INTRODUCTION: APTO-253 has captured attention because of its distinguishing mechanism of action as the only known a small molecule in clinical development to target a conserved structure in the promoter of the MYC oncogene and interrupt MYC gene expression. In preclinical studies of acute myeloid leukemia (AML) cell lines, APTO-253 was discovered to potently down-regulate MYC gene expression, reduce MYC mRNA and protein level, induce apoptosis, and deplete cells of the MYC oncoprotein. APTO-253 demonstrated broad killing of primary mononuclear cells isolated from the bone marrow of patients with AML, MDS or MPN and enhanced AML cell killing when combined with BET bromodomain inhibitors or FLT3 inhibitors (Kurtz, Blood 2017 126:1358). Because dysregulated MYC is considered a major oncogenic operator in AML and myelodysplasias and because APTO-253 has such a distinct mechanism to kill such cells, APTO-253 has been granted orphan drug designation for the treatment of AML by the US FDA and is currently in a Phase 1a/b clinical trial in patients with relapsed or refractory AML (R/R AML) or high-risk myelodysplasias (high-risk MDS). METHODS: APTO-253 is being evaluate in an ongoing clinical trial, entitled "A Phase I a/b Dose Escalation and Expansion, Multicenter, Open-label, Safety, Pharmacokinetic and Pharmacodynamic Study of APTO-253 in Patients with Relapsed or Refractory Acute Myelogenous Leukemia or High-Risk Myelodysplasia (NCT02267863)". The protocol provides for patients to be dosed once weekly on days 1, 8, 15, and 22 of each 28-day cycle with the intended dose levels of 20, 40, 66, 100, 140, 180 or 220 mg/m2. Eligible patients must be ≥18 years old, have a life expectancy of at least 2 months, and have acceptable hematologic, renal and liver functions and coagulation status parameters. Primary objectives are to determine the safety and tolerability of APTO-253, to determine the maximum tolerated dose (MTD) and the dose limiting toxicities (DLT), and to establish the recommended Phase 2 dose for patients with specific types of hematologic malignancies. Key secondary objectives are to seek evidence of antitumor activity by hematologic and bone marrow evaluations and to assess the impact of APTO-253 on the expression of pharmacodynamic biomarkers (including MYC and p21 gene expression). RESULTS: To date, R/R AML and high-risk MDS patients have been enrolled and treated at dose levels of 20, 40 and 66 mg/m2 APTO-253. As specified by the protocol, only one patient was required at each of the 20 and 40 mg/m2 doses. The AML patient on the lowest dose level completed the 28-day cycle and demonstrated approximately a 72% reduction in MYC expression levels in peripheral white blood cells during the cycle. The high-risk MDS patient who received 40 mg/m2 demonstrated a 79% reduction in MYC expression during the first cycle. To date (as of July 2019), no drug related TEAEs, SAEs or dose limiting toxicities have been reported. Two patients are receiving treatment with 66 mg/m2, and samples are being analyzed for a series of pharmacodynamic markers. CONCLUSIONS: APTO-253 is a potent molecule with a particularly interesting and novel mechanism of action that results in robust suppression of MYC expression. The Phase 1 a/b trial has successfully enrolled R/R-AML and HR-MDS patients into the first three cohorts at 20, 40 and 66 mg/m2. Clinical data to date suggest APTO-253 is generally well tolerated at the doses tested, and target engagement has been evidenced by the reduction in cellular MYC gene expression in whole blood samples from R/R AML and high-risk MDS patients. Recruitment and enrollment are continuing, and updated safety, PK and biomarker data will be presented at the meeting. Disclosures Howell: Aptose Biosciences, Inc: Consultancy, Research Funding. Zhang:Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Targeted Killing of AML Cells By Bispecific Antibody Armed Activated T Cells

Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of hematopoietic stem/progenitor cells that poses a significant therapeutic challenge. Recurrence of the disease is attributed to leukemia-initiating cells, also referred to as leukemic stem cells (LSCs), which are thought to be spared from chemotherapy and capable of reinitiating the disease. Thus, novel non-toxic therapeutic strategies to target and eradicate LSCs and AML blasts are urgently needed. Immunotherapy (IT) is an attractive strategy to improve outcomes for patients with AML, as it does not rely on the cytotoxic mechanisms employed by conventional chemotherapies. Methods: Bispecific antibody was produced by chemical hetero-conjugation of anti-CD33 monoclonal antibody (gemtuzumab ozogamicin [GO]) linked to calicheamicin to anti-CD3 monoclonal antibody (anti-CD3 x GO bispecific antibody, CD33Bi) or anti-CD3 and anti-CD123 (CD123Bi). We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD33Bi and CD123Bi armed ATC using flow cytometry-based cytotoxicity assays. Cytokines and chemokines released during CD33Bi or CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. A xenogeneic NOD/SCID/gamma chain KO (NSG) mouse model was used for the evaluation of in vivo activity of Bispecific antibody (50ng/million cells) Armed activated T cells (BATs). Engraftment of AML cells-KG1 was tracked by quantifying the human CD45+ AML cells using flow cytometry. Two patient samples were analyzed for blast and LSC population before and after incubation with CD123-BATs. Results: In a non-radioactive quantitative flow cytometry-based cytotoxicity assay Bispecific antibody (50ng/million cells) Armed activated T cells (BATs) show substantial killing by both CD33-BATs and CD123-BATs against AML cell lines (EOL1, KG1, NoMo1 and TF-1) at 1:1 and 2:1 E/T ratios ranging from 60-90% cytotoxicity and produced increased levels of Th1 cytokines (TNF-alpha, GM-CSF and IFN-gamma). The KG1 engrafted 6- to 8-week-old NSG mice (n=8 mice/group) injected IV with 20 x 106 CD33-BATs (3x/week for 4 weeks; 100ml/injection) was able to inhibit leukemic cell expansion significantly compared to 0.06 mg/kg gemtuzumab ozogamicin [GO] antibody (p<0.0006); likewise significant inhibition of leukemic cell expansion was observed with CD123-BATs compared to PBS control mice (p<0.009). One patient sample incubated with CD123-BATs showed significant reduction in blast after CD123-BATs treatment compared to before treatment (13.2% vs. 30%) or CD34+CD38- LSC after CD123-BATs treatment (5.2%) compared to before treatment (20%). Second patient sample also showed 50% reduction in blast population, no LSC were detected in this sample. Conclusions: ATC armed with CD33Bi or CD123Bi at 50 ng/106 cells showed comparable cytotoxicity directed at either high or low leukemic blast populations, able to inhibit leukemic cell expansion significantly compared to the control mice. This approach may provide a potent and non-toxic strategy to target leukemic blasts and leukemic stem cells. Disclosures Huang: TransTarget Inc.: Other: Co-Founder.

Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in Combination with Decitabine in Patients with High-Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in Combination with Decitabine in Patients with High-Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

A Sampling of Highlights from the Literature

New clonotypes recruited (by Staff Sergeant K. Brasier via U.S. Air Force)To identify which tumor-specific T cells are active after anti–PD-1 therapy of basal cell carcinomas, single cells from biopsies before and after treatment were analyzed for gene expression and TCR usage. T cells already exhausted before anti–PD-1 therapy were not reactivated. Instead, novel clonotypes (only a small fraction of which had previously expressed TCF7) replaced those, transiting from memory to activated states. This was also true of squamous cell carcinoma, suggesting that checkpoint blockade prompts a new wave of immunoevolution and that the tumor's ability to constantly attract new T cells may be key to successful responses.Yost KE, …, Chang HY. Nat Med 2019 Aug 1;25:1251–9.CD163+ TAMs infiltrating melanomas (from Fig. 1C Etzerodt et al. J Exp Med 2019)Tumor-associated macrophages (TAMs) can contribute to tumor cell immune evasion, which is dependent on their phenotype and the tumor microenvironment (TME). Barkal et al. show that ovarian and breast cancers overexpress CD24, which relays “don't eat me” signals to TAMs via interaction with Siglec-10. Etzerodt et al. demonstrate that in melanomas refractory to anti–PD-1, CD163+ TAMs are elevated. Immunotherapies that target either surface molecules (such as CD24) or cell types (such as CD163+ TAMs) that promote immune evasion can alter the TME, resulting in enhanced antitumor immunity.Barkal AA, …, Weissman IL. Nature 2019 Aug 15;572:392–6.Etzerodt A, …, Lawrence T. J Exp Med 2019 Aug 2. DOI: 10.1084/jem.20182124.Optimizing the anti–PD1 schedule (by Best Picko via Flickr)Optimizing the efficacy of combination therapies involves determining their ideal schedule. In anti–PD-1–resistant murine tumor models, peptide vaccination plus anti–PD-1 slows tumor growth, whereas exposure to anti–PD-1 before combination treatment abrogates effectiveness. Anti–PD-1 causes suboptimal antigen priming of intratumoral CD8+ T cells, leading to increased dysfunction, higher PD-1+ CD38hi frequency, and more death. Anti–PD-1 responder patients have fewer intratumoral PD-1+ CD38+ CD8+ T cells compared to nonresponders, suggesting a predictive biomarker for anti–PD-1. Thus, T-cell priming prior to anti–PD-1 therapy is crucial for its efficacy.Verma V, …, Khleif SN. Nat Immunol 2019 Sep 1;20:1231–43.IFN opposing itself (via Max Pixel)Resistance to immune checkpoint blockade (ICB) is complex. Tumor-produced IFNγ sets up feedback loops that promote intrinsic tumor resistance and inhibit innate and adaptive immunity. Blocking IFN-stimulated gene expression from MHC class Ilow tumors can restore ICB responsiveness, stimulating tumor killing via TRAIL by NK and type 1 innate lymphoid cells (ILC1s). ICB treatment enhances IFNγ produced by CD8+ exhausted T (TEX) cells (that had been inactivated through PD-1 engagement), which supports NK/ILC1 cell maturation; TEX cells can also kill those tumor cells with adequate MHC class I, via the granzyme B/perforin pathway.Benci JL, …, Minn AJ. Cell 2019 Aug 8;178:933–48.e14.Engineering increased functionality (by N. Pawar via MotorsGarage)The affinity of a TCR for its target can affect antileukemia responses in patients with acute myeloid leukemia (AML) treated by hematopoietic cell transplantation (HCT). Engineered T cells bearing a high-affinity TCR targeting antigen WT1 (called TCRC4) can kill AML cells, are polyfunctional, and have increased persistence in vivo. When infused into patients after HCT, TCRC4 T cells reduced AML relapse. Thus, these engineered T cells could extend antileukemia T-cell persistence and reduce relapse in high-risk AML patients after HCT or other standards of care.Chapuis AG, …, Greenberg PD. Nat Med 2019 Jul 3;25:1064–72.Conditioning of hosts (by didkog via Pixabay)The efficacy of adoptive cell therapy (ACT) for cancer depends on prior lymphodepletion of recipients: IL-1β increases after this “conditioning.” Adding IL-1β to ACT therapy greatly improves therapeutic responses by increasing the number of effector CD8+ T cells in tumors and tumor-draining lymph nodes compared to ACT alone, in an IL-2/IL-15–mediated manner. IL-1β improves the trafficking of CD8+ T cells into peripheral organs and primes the host to better support effector CD8+ T-cell survival in these organs. Thus, treating patients with IL-1β may improve ACT efficacy.Lee P-H, …, Restifo NP. J Exp Med 2019 Aug 12. DOI: 10.1084/jem.20181218.

PF207 TARGETING WILMS TUMOR 1 WITH A T CELL BISPECIFIC ANTIBODY (WT1‐TCB): EX VIVO AND IN VIVO POTENCY BY BIVALENT RECOGNITION OF PEPTIDE‐MHC COMPLEXES FROM AN INTRACELLULAR TUMOR ANTIGEN

Background:Antibody‐based immunotherapy represents a promising strategy to target chemo‐resistant leukemic cells. However, classical antibody‐based approaches are restricted to cell surface antigens, even though targeting of intracellular antigens would allow further access to a large number of tumor‐specific target structures.Aims:In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody based on T cell receptor (TCR)‐like antibodies for immunotherapy of acute myeloid leukemia (AML). The TCB targets the intracellular tumor antigen Wilms Tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A∗02:01 (HLA‐A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR‐specificity.Methods:WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real‐time PCR, western blot and immunohistochemical staining. WT1‐TCB‐mediated cytotoxicity was analyzed by co‐cultivation of WT1‐expressing HLA‐A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCB‐mediated cytotoxicity against primary AML cells was evaluated in our pre‐established feeder layer‐based ex vivo long‐term culture system. For in vivo testing, NSG mice (NOD.Cg‐Prkdcscid‐Il2rgtm1Wjl/SzJ) were humanized with human HLA‐A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1‐expressing HLA‐A2+ SKM‐1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1‐TCB.Results:In accordance with previous reports, we observed WT1 expression in 79% (n = 38) of cancer cell lines and in 92% (n = 65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n = 26), whereas WT1 was expressed again at time of relapse (n = 21). WT1‐TCBs elicited antibody‐mediated T cell cytotoxicity against peptide‐pulsed T2 cells and AML cell lines in a WT1 and HLA‐restricted manner. Equally, TCR downstream signaling was observed in a WT1‐restrictive manner by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67 ± 6% after 13–14 days; ± SEM; n = 18). Correspondingly, up‐regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3 ± 1.5, PD‐1: 5.1 ± 0.7, TIM‐3: 4.7 ± 0.6; ± SEM; n = 22). WT1‐TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38 ± 13% after 13–14 days; ± SEM; n = 5). In comparison with WT1RMF‐specific T cells, only bivalent binding by WT1‐TCB induced efficient lysis of primary AML cells. Furthermore, WT1‐TCB‐treated humanized mice bearing SKM‐1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control.Summary/Conclusion:TCR‐like TCBs targeting of intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide‐MHC complex on the cell surface validating WT1‐TCB as a promising therapeutic agent for the treatment of AML.

Synergistic Cytotoxicity of Methyl 4-Hydroxycinnamate and Carnosic Acid to Acute Myeloid Leukemia Cells via Calcium-Dependent Apoptosis Induction.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor prognosis for most patients. Conventional chemotherapy has been the standard treatment approach for AML in the past 40 years with limited success. Although, several targeted drugs were recently approved, their long-term impact on survival of patients with AML is yet to be determined. Thus, it is still necessary to develop alternative therapeutic approaches for this disease. We have previously shown a marked synergistic anti-leukemic effect of two polyphenols, curcumin (CUR) and carnosic acid (CA), on AML cells in-vitro and in-vivo. In this study, we identified another phenolic compound, methyl 4-hydroxycinnamate (MHC), which among several tested phytochemicals could uniquely cooperate with CA in killing AML cells, but not normal peripheral blood mononuclear cells. Notably, our data revealed striking phenotypical and mechanistic similarities in the apoptotic effects of MHC+CA and CUR+CA on AML cells. Yet, we show that MHC is a non-fluorescent molecule, which is an important technical advantage over CUR that can interfere in various fluorescence-based assays. Collectively, we demonstrated for the first time the antileukemic activity of MHC in combination with another phenolic compound. This type of synergistically acting combinations may represent prototypes for novel antileukemic therapy.

T-cell Activity against AML Improved by Dual-Targeted T Cells Stimulated through T-cell and IL7 Receptors.

The development of engineered T cells to treat acute myeloid leukemia (AML) is challenging due to difficulty in target selection and the need for robust T-cell expansion and persistence. We designed a T cell stimulated to kill AML cells based on recognition of the AML-associated surface marker CLEC12A, via secretion of a CLEC12AxCD3 bispecific "engager" molecule (CLEC12A-ENG). CLEC12A-ENG T cells are specifically activated by CLEC12A, are not toxic to hematopoietic progenitor cells, and exhibit antigen-dependent AML killing. Next, we coupled stimulation of T-cell survival to triggering of a chimeric IL7 receptor with an ectodomain that binds a second AML-associated surface antigen, CD123. The resulting T cells, identified as CLEC12A-ENG.CD123IL7Rα T cells, demonstrate improved activation upon dual target recognition, kill AML, and exhibit antitumor activity in xenograft models. Enhanced T-cell activation conferred by CD123.IL7Rα was dependent both on recognition of the CD123 target and on IL7Rα-mediated downstream signaling. Expression of a chimeric IL7R targeted to a second tumor-associated antigen (TAA) should improve T-cell activity not only against hematologic malignancies, but perhaps against all cancers.

NF-κB and Poly (ADP-ribose) Polymerase 1 Form a Positive Feedback Loop that Regulates DNA Repair in Acute Myeloid Leukemia Cells.

NF-κB mediates acquired resistance in acute myeloid leukemia (AML) cells treated with DNA-damaging agents. Because DNA repair is the major molecular shift that alters sensitivity to DNA-damaging agents, we explored whether activation of the NF-κB pathway promotes AML cell survival by regulating DNA repair after chemotherapy. Our results showed that RELA, an important subunit of NF-κB, regulated DNA repair by binding to the promoter region of the PARP1 gene and affecting PARP1 gene transcription. Conversely, PARP1 knockdown reduced NF-κB activity, indicating that NF-κB and PARP1 create a positive feedback loop in DNA repair. Simultaneous treatment with the NF-κB inhibitor BMS-345541 and the PARP1 inhibitor olaparib resulted in robust killing of AML cells. This dual inhibition significantly suppressed tumor growth and extended survival times in xenograft tumor models. IMPLICATIONS: RELA and PARP1 form a positive feedback loop to regulate DNA damage repair, simultaneous inhibition of NF-κB and PARP1 increases the antileukemic efficacy of daunorubicin in vitro and in vivo, broadening the use of PARP1 inhibitors.

Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia

GSK3α has been identified as a new target in the treatment of acute myeloid leukemia (AML). However, most GSK3 inhibitors lack specificity for GSK3α over GSK3β and other kinases. We have previously shown in lung cancer cells that GSK3α and to a lesser extent GSK3β are inhibited by the advanced clinical candidate tivantinib (ARQ197), which was designed as a MET inhibitor. Thus, we hypothesized that tivantinib would be an effective therapy for the treatment of AML. Here, we show that tivantinib has potent anticancer activity across several AML cell lines and primary patient cells. Tivantinib strongly induced apoptosis, differentiation and G2/M cell cycle arrest and caused less undesirable stabilization of β-catenin compared to the pan-GSK3 inhibitor LiCl. Subsequent drug combination studies identified the BCL-2 inhibitor ABT-199 to synergize with tivantinib while cytarabine combination with tivantinib was antagonistic. Interestingly, the addition of ABT-199 to tivantinib completely abrogated tivantinib induced β-catenin stabilization. Tivantinib alone, or in combination with ABT-199, downregulated anti-apoptotic MCL-1 and BCL-XL levels, which likely contribute to the observed synergy. Importantly, tivantinib as single agent or in combination with ABT-199 significantly inhibited the colony forming capacity of primary patient AML bone marrow mononuclear cells. In summary, tivantinib is a novel GSK3α/β inhibitor that potently kills AML cells and tivantinib single agent or combination therapy with ABT-199 may represent attractive new therapeutic opportunities for AML.