Target Acute Myeloid Leukemia Cells Research Articles

8-Cl-Ado and 8-NH2-Ado synergize with venetoclax to target the methionine-MAT2A-SAM axis in acute myeloid leukemia

Targeting the metabolic dependencies of acute myeloid leukemia (AML) cells is a promising therapeutical strategy. In particular, the cysteine and methionine metabolism pathway (C/M) is significantly altered in AML cells compared to healthy blood cells. Moreover, methionine has been identified as one of the dominant amino acid dependencies of AML cells. Through RNA-seq, we found that the two nucleoside analogs 8-chloro-adenosine (8CA) and 8-amino-adenosine (8AA) significantly suppress the C/M pathway in AML cells, and methionine-adenosyltransferase-2A (MAT2A) is one of most significantly downregulated genes. Additionally, mass spectrometry analysis revealed that Venetoclax (VEN), a BCL-2 inhibitor recently approved by the FDA for AML treatment, significantly decreases the intracellular level of methionine in AML cells. Based on these findings, we hypothesized that combining 8CA or 8AA with VEN can efficiently target the Methionine-MAT2A-S-adenosyl-methionine (SAM) axis in AML. Our results demonstrate that VEN and 8CA/8AA synergistically decrease the SAM biosynthesis and effectively target AML cells both in vivo and in vitro. These findings suggest the promising potential of combining 8CA/8AA and VEN for AML treatment by inhibiting Methionine-MAT2A-SAM axis and provide a strong rationale for our recently activated clinical trial.

PIEZO1 is essential for the survival and proliferation of acute myeloid leukemia cells.

Leukemogenesis is a complex process that interconnects tumoral cells with their microenvironment, but the effect of mechanosensing in acute myeloid leukemia (AML) blasts is poorly known. PIEZO1 perceives and transmits the constraints of the environment to human cells by acting as a non-selective calcium channel, but very little is known about its role in leukemogenesis. For the first time, we show that PIEZO1 is preferentially expressed in healthy hematopoietic stem and progenitor cells in human hematopoiesis, and globally overexpressed in AML cells. In AML subtypes, PIEZO1 expression associates with favorable outcomes as better overall (OS) and disease-free survival (DFS). If PIEZO1 is expressed and functional in THP1 leukemic myeloid cell line, its chemical activation doesn't impact the proliferation, differentiation, nor survival of cells. However, the downregulation of PIEZO1 expression dramatically reduces the proliferation and the survival of THP1 cells. We show that PIEZO1 knock-down blocks the cell cycle in G0/G1 phases of AML cells, impairs the DNA damage response pathways, and critically increases cell death by triggering extrinsic apoptosis pathways. Altogether, our results reveal a new role for PIEZO1 mechanosensing in the survival and proliferation of leukemic blasts, which could pave the way for new therapeutic strategies to target AML cells.

Targeting NRF2 uncovered an intrinsic susceptibility of acute myeloid leukemia cells to ferroptosis

Drug resistance and poor treatment response are major obstacles to the effective treatment of acute myeloid leukemia (AML). A deeper understanding of the mechanisms regulating drug resistance and response genes in AML is therefore urgently needed. Our previous research has highlighted the important role of nuclear factor E2-related factor 2 (NRF2) in AML, where it plays a critical role in detoxifying reactive oxygen species and influencing sensitivity to chemotherapy. In this study, we identify a core set of direct NRF2 targets that are involved in ferroptosis, a novel form of cell death. Of particular interest, we find that glutathione peroxidase 4 (GPX4) is a key ferroptosis gene that is consistently upregulated in AML, and high expression of GPX4 is associated with poor prognosis for AML patients. Importantly, simultaneous inhibition of NRF2 with ML385 and GPX4 with FIN56 or RSL3 synergistically targets AML cells, triggering ferroptosis. Treatment with ML385 + FIN56/RSL3 resulted in a marked reduction in NRF2 and GPX4 expression. Furthermore, NRF2 knockdown enhanced the sensitivity of AML cells to the ferroptosis inducers. Taken together, our results suggest that combination therapy targeting both NRF2 and GPX4 may represent a promising approach for the treatment of AML.

KLF4-mediated upregulation of the NKG2D ligand MICA in acute myeloid leukemia: a novel therapeutic target identified by enChIP

The immunoreceptor NKG2D, which is expressed on NK cells and T cell subsets is critically involved in tumor immune surveillance. This applies in particular to acute myeloid leukemia (AML), which evades immune detection by downregulation of NKG2D ligands (NKG2D-L), including MICA. The absence of NKG2D-L on AML cells is moreover associated with leukemia stem cell characteristics. The NKG2D/NKG2D-L system thus qualifies as an interesting and promising therapeutic target.Here we aimed to identify transcription factors susceptible to pharmacological stimulation resulting in the expression of the NKG2D-L MICA in AML cells to restore anti-tumor activity. Using a CRISPR-based engineered ChIP (enChIP) assay for the MICA promoter region and readout by mass spectrometry-based proteomics, we identified the transcription factor krüppel-like factor 4 (KLF4) as associated with the promoter. We demonstrated that the MICA promoter comprises functional binding sites for KLF4 and genetic as well as pharmacological gain- and loss-of-function experiments revealed inducible MICA expression to be mediated by KLF4.Furthermore, induction in AML cells was achieved with the small compound APTO253, a KLF4 activator, which also inhibits MYC expression and causes DNA damage. This induction in turn yielded increased expression and cell surface presentation of MICA, thus rendering AML cells more susceptible to NK cell-mediated killing. These data unravel a novel link between APTO253 and the innate anti-tumor immune response providing a rationale for targeting AML cells via APTO253-dependent KFL4/MICA induction to allow elimination by endogenous or transplanted NK and T cells in vivo.D1rDmzAPriWUyYABRWPo9PVideo

Dual IKZF2 and CK1α degrader targets acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Synthetic Lipid Nanoparticles Actively Target Acute Myeloid Leukemia (AML) Cells and Induce Ferroptosis through Decreased Expression of Glutathione Peroxidase 4

Introduction: Relapsed and refractory AML patients have a 30% 5-year overall survival (SEER 2012-2018). We found that certain cancer cells that are highly dependent on cholesterol uptake and express the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), scavenger receptor type B-1 (SR-B1), also highly express the redox enzyme, glutathione peroxidase 4 (GPX4) (Rink et al 2020). GPX4 has been shown to detoxify lipid peroxides in cancer cells and prevent cell death through ferroptosis, an iron-dependent redox form of cell death. Using a synthetic lipid nanoparticle (Au-LNP) that actively targets SR-B1, we showed modulation of cancer cell cholesterol uptake and metabolism is accompanied by a reduction in GPX4 expression. As a result, Au-LNP therapy kills these cells via ferroptosis. In AML, high GPX4 expression is associated with worse overall survival (Akiyama et al. Blood 2021). Therefore, we hypothesized that high SR-B1 expression would enable Au-LNP to target AML cells, modulate cell cholesterol, and reduce GPX4 resulting in AML cell death by ferroptosis. Methods: GEPIA is an interactive web server that houses the RNA sequencing expression data from the TCGA and the GTEx projects. We selected the LAML database and interrogated the survival data using the top 25% and bottom 25% SR-B1 expression as cutoffs. The cell lines MV4-11 (biphenotypic B-myelomonocytic AML with translocation t(4;11) and FLT3-ITD mutation), HEL (erythroblast AML), and U937 (promonocytic AML) were used for these studies. Au-LNPs were synthesized by surface-functionalizing 5nm gold (Au) nanoparticles with apolipoprotein A-I and phospholipids. SR-B1 and GPX4 levels were measured by western blot and flow cytometry. Cell viability was measured using the MTS assay, annexin V/propidium iodide staining, and a colony forming assay. Colonies were counted by an automated microscope and analyzed with Fiji. Lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay. Surface biomarker changes were measured by flow cytometry. Primary AML samples were obtained by collecting the buffy coat from peripheral blood samples from 2 patients with AML and isolating CD34 positive blasts (IRB# STU00208941). Results: In the LAML database in GEPIA, we found a significant survival detriment with high SR-B1 expression (75th percentile) when compared with low SR-B1 expression (25th percentile; p = 0.023; hazard ratio = 2.5) (Figure 1). SR-B1 expression was observed in all three AML cell lines tested (MV4-11, HEL, U937). Au-LNPs potently induced cell death in each cell line, with IC50s for MV4-11 (4.7 nM), HEL (0.8 nM), and U937 (4.6 nM) that were much lower than with cytarabine (535 nM, 259 nM, and 96 nM, respectively). In the colony forming assays, Au-LNPs significantly decreased MV4-11 colony counts compared with PBS control. By contrast, β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) inhibitors, such as atorvastatin and rosuvastatin, had no effect on cell viability, with concentrations up to 50 µM. Au-LNPs reduced GPX4 expression in MV4-11 and HEL cells, leading to the accumulation of lipid peroxides by C11 BODIPY assay. Au-LNPs-induced cell death was prevented by the addition of the lipophilic antioxidant ferrostatin-1 and the iron chelator deferoxamine (DFO), supporting the hypothesis that Au-LNP-induced cell death occurred by ferroptosis. Low dose Au-LNPs increased expression of CD11c (dendritic cell marker), CD16 (monocyte marker), and CD24 (neutrophil marker) on MV4-11 cells, suggesting an increase in myeloid differentiation. Finally, we isolated CD34+ blasts from peripheral blood samples of two AML patients. Both patients’ blasts expressed SR-B1 as detected by flow cytometry, and Au-LNPs showed significant cell killing in both patient samples. Conclusion: We have demonstrated that Au-LNP target SR-B1, expressed by both AML cell lines and in primary AML samples, and induce cell death by ferroptosis resulting from depletion of GPX4. Future studies targeting the cholesterol scavenger receptor SR-B1 and investigating relevant resistance pathways and opportunities for synergy with available agents are indicated. Figure 1. Overall survival of AML patients in the LAML database stratified by scavenger receptor B1 expression. (Tang, Z. et al. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res, 10.1093/nar/gkx247) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MP0533: A Multispecific Darpin CD3 Engager Targeting CD33, CD123, and CD70 for the Treatment of AML and MDS Designed to Selectively Target Leukemic Stem Cells

Introduction: Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse. Newer therapies, including chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) that target specific tumor associated antigens (TAAs) overexpressed on stem cells and leukemic blasts, are promising options for AML. We postulated that a TCE able to simultaneously target multiple TAAs could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while minimally affecting healthy cells. Moreover, the simultaneous targeting of different TAAs has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each TAA. Methods: Our DARPin (Designed Ankyrin Repeat Protein) libraries contain trillions of molecules allowing the generation of highly diverse binders against target proteins that can be easily combined into multi-specific DARPins to elicit desired biological effects. We leveraged this proprietary platform to screen multi-specific CD3 engaging DARPin molecules, including serum albumin binding DARPins for systemic half-life extension, to generate MP0533, the first half-life extended avidity-engineered CD3 engaging DARPin capable of targeting CD33, CD123, and CD70 simultaneously. Efficacy and safety of MP0533 was extensively tested in vitro and ex vivo, with cell lines and primary cells in both allogenic and autologous settings, and in vivo in different cell line-derived xenograft mouse models. Results: We demonstrated the avidity effect of the multi-targeting MP0533 in co-culture assays with genetically engineered Molm-13 cells and allogenic T cells, where MP0533 induced up to 10-fold higher T cell mediated killing of cells expressing any combination of at least 2 of the 3 targeted TAAs vs. cells expressing a single TAA. Overall, an EC50 potency ranging from 5 to 25 pM was measured on multiple cell lines showing different levels of expression and co-expression of all TAAs (Molm-13, THP-1, RPMI8226). Efficacy was additionally assessed on primary unsorted AML bone marrow samples: MP0533 demonstrated the ability to induce autologous T cell activation and killing of AML cells in samples from newly diagnosed and previously treated patients with different TAA expression/co-expression levels and E:T ratios ranging from 10:1 to 1:28. MP0533-induced killing of LSC and selectivity over healthy hematopoietic stem cells (HSCs) was analyzed in clonogenic assays in co-cultures of sorted CD34+ cells with allogenic T cells. After 4 days initial killing assay, and 2 weeks colony culture in semi-solid media, MP0533 was able to show depletion of LSC at doses where HSC where only minimally affected. While T-cell activation, proliferation, and cytotoxicity were not affected, the multi-specific TCE format of MP0533 resulted in a significant decrease in cytokine release in both tumor cell killing assays and whole blood test systems when compared to other mono-targeting TCEs, indicating potential for improved safety. Moreover, the avidity approach and the safer profile on single-TAA expressing cells was confirmed by the absence of toxicity measured on CD123-expressing endothelial cells, indicating a low risk of capillary leak syndrome induced by MP0533. Also, potential T cell fratricide induced by CD70 expression on activated T cells was not observed. Finally, efficacy and safety profiles were also confirmed in in vivo xenograft models in human PBMC- or CD34-reconstituted mice. In such models MP0533 induced T cell infiltration in s.c. implanted tumors, leading to anti-tumor activity. Importantly, activation of T cells and release of cytokines were only localized in the tumors, indicating absence of potential systemic on-target off-tumor toxicities induced by MP0533. Conclusion: We generated a multi-specific avidity-driven CD3 engaging DARPin molecule with tailored affinities towards different TAAs showing high efficacy and with the potential for better LSC selectivity and lower cytokine release compared with mono-specific TCE approaches. Further, our approach to achieve systemic half-life extension could ensure a more convenient Q1W IV therapy. A Phase I clinical trial to evaluate safety and dose of MP0533 in humans is imminent.

Generation Natural Killer Cell-Mimic Nanoparticles for Active Targeting of Acute Myeloid Leukemia

Natural killer (NK) cells play a crucial role in recognizing and killing emerging tumor cells. Via an array of activating and inhibitory receptors, NK cells can target and kill abnormal cells, including tumor cells, without prior antigen sensitization. However, fully-established tumors employ numerous mechanisms to inactivate NK cells or hide from them, for example by shedding NK cell-activating ligands, like NK2GD (Chiossone et al., 2018). Here, we describe the engineering of nanoparticles (NPs) functionalized with tumor-recognizing and cytotoxic components of NK cells to address this problem and develop a nanoparticle-based treatment strategy simulating NK cell functionality. Liposomes were synthesized using the thin film hydration method followed by extrusion and functionalized them with the death ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), immunoglobulin Fc-binding peptide (FCP, replicating the function of NK CD16 receptor) and the therapeutic antibody, anti-CD38 (daratumumab, Panel A) to target acute myeloid leukemia (AML). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to monitor morphology and size of liposomes. The potential of NK cell-mimic NPs (CD38-NK.NPs) to target and kill AML cells was evaluated on primary AML blasts and a disseminated AML xenograft for which the CD38-positive AML cell line, OCI-AML2 was engrafted into 8-9 week-old female NSG mice. Upon establishment of AML, the mice were injected intraperitoneally (i.p) with 5 doses of either naked liposomes (as vehicle control), soluble TRAIL protein (sTRAIL), LP/TRAIL or CD38-NK.NPs every two days. Tumor burden was monitored by determining the frequency of live human CD45+ cells in the peripheral blood, spleen and bone marrow with flow cytometry. Liposome was chosen as the base of the NP due to its high biocompatibility, biodegradability, cell-like characteristics and capability to penetrate through vessel walls (Pattni, Chupin and Torchilin, 2015). TEM and DLS measurements showed the generated NK.NPs were monodisperse with an average diameter of 145 nm, a size enabling vessel transmigration and avoiding kidney filtration (Gaumet et al., 2008). Immunofluorescent labelling of TRAIL, FCP and antibody verified CD38-NK.NPs functionalization. In vitro, both TRAIL-conjugated liposomes and CD38-NK.NPs were more effective in killing AML cell lines (ML-1, ML-2, Kasumi-1, and OCI-AML2) than sTRAIL, indicating that TRAIL-mediated cytotoxicity can be enhanced by presenting TRAIL in a conformation close to its membrane-bound structure. No significant difference could be detected between LP/TRAIL vs. CD38-NK.NP cytotoxicity in the in vitro assays. On the other hand, CD38-NK.NPs were more effective in killing primary AML blasts than non-targeted, NP-conjugated TRAIL (LP/TRAIL, 13±17% vs 4±15% killing, respectively). Moreover, CD38-NK.NPs showed considerably higher activity relative to the NK cell line, KHYG-1, against CD38-positive AML cell lines. Notably, CD38-NK.NPs showed no significant cytotoxic effect on non-malignant peripheral blood leukocytes. In vivo, NK cell-mimic NPs were more efficient in targeting and eradicating CD38-positive AML cells that either sTRAIL or LP/TRAIL, shown by the reduced percentage of circulating hCD45+ AML cells in treated mice (Panel B). CD38-NK.NPs were also more efficient in inhibiting accumulation of AML cells in the bone and spleen than sTRAIL and LP/TRAIL (Panel B), confirming the ability of CD38-NK.NPs to actively target AML cells expressing the targeted biomarker. Of note, CD38-NK.NPs showed no detectable systemic toxicity or damage to kidneys, liver and pancreas in healthy NSG mice. Overall, we have developed an NK cell-mimic nanoparticle able to actively target AML based on AML surface marker expression without any detectable systematic toxicity. Importantly, due to the modular built of the NK.NPs, they can be functionalized by any therapeutic tumor-targeting antibody via the immunoglobulin-binding peptide (FCP), thus enabling selective targeting tumor cells based on expression of tumor-specific markers and enabling personalized therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ligand-based discovery of a novel GATA2 inhibitor targeting acute myeloid leukemia cells

Despite major therapeutic advances leading to improved patient outcomes for other haematological malignancies, development of new therapeutics to improve prognosis for acute myeloid leukemia (AML) patients remains an area of unmet clinical need. Overexpression of GATA2, a member of the GATA family of zinc finger transcription factors, has been implicated in AML. In settings where GATA2 is overexpressed in human AML cells, K7174, a proteasome inhibitor that inhibits GATA2, induces apoptosis and enhances the killing activity of AML chemotherapeutics in vitro yet targeting the proteasome has been associated with high toxicity in the clinic. Using an in silico approach, we embarked on a screen to identify specific GATA2 inhibitors that will target AML cells independently of the proteasome. A shape-based virtual screening of an in-house library of small molecules was performed using a low-energy conformation of K7174. The virtual hit compounds were subsequently filtered according to their potential selectivity for GATA2 over the proteasome. From 15 selected compounds evaluated for their ability to kill AML cells in vitro, one compound, an asymmetrical substituted piperazine with Hepatitis C antiviral activity, exhibited superior ability to induce apoptosis and reduce cell cycling in AML cells without proteasome inhibition. This compound was also able to promote cell death of the relapse propagating leukemic stem cell (LSC) compartment while sparing Gata2 knockout LSCs, crucially demonstrating specificity to inhibit GATA2. We have identified a GATA2 specific inhibitor with promising capability to target AML cells in vitro, including LSCs that underpin poor prognosis in AML.

Synergy of Venetoclax and 8-Chloro-Adenosine in AML: The Interplay of rRNA Inhibition and Fatty Acid Metabolism.

Simple SummaryTreatment failures of acute myeloid leukemia (AML) have been attributed to the persistence of leukemia stem cells (LSCs), which are refractory to conventional treatments. Venetoclax (VEN), currently FDA-approved in combination with low-dose cytarabine or hypomethylating agents, is highly effective in inducing disease remission in patients with de novo AML; however, most of these patients eventually relapse; thus, novel VEN combinations are urgently needed. In this regard, 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets AML cells, including LSCs. We demonstrate that VEN and 8-Cl-Ado cooperate in targeting ribosomal RNA synthesis and mitochondrial metabolism in LSCs, thereby decreasing LSC survival. Given the emerging concept that LSC behavior is strongly associated with protein synthesis regulation and mitochondrial metabolism, our results suggest that the VEN/8-Cl-Ado combination is a promising regimen for the treatment of patients with relapsed AML.It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.

3022 – METABOLIC AND EPIGENETIC IMPACT OF CD44 ANTIBODY-INDUCED DIFFERENTIATION IMPLICATES THE USE OF EPIGENETIC THERAPIES IN AML TREATMENT

Acute myeloid leukemia (AML) is a subset of leukemia characterized by the clonal expansion of abnormal myeloid blasts. The poor prognosis of AML patients caused by low tolerance to intensive chemotherapy and high relapse rates triggered by leukemic stem cells has encouraged the pursuit of alternative therapies. Differentiation therapy, which involves the use of agents that can release the differentiation block in these leukemic stem/blasts has emerged as a promising therapy following the success of All-trans retinoic acid (ATRA) on one AML subtype. Efforts focused on finding differentiation agents that are effective on other AML subtypes brought into light the possibility of using anti-CD44 antibodies to target AML cells. Anti-CD44 has been shown to release the differentiation block in varying AML subtypes and work in our lab is focused on understanding the mechanism by which it induces its effects. Recent studies highlight the role that epigenetic and metabolic regulation plays during hematopoiesis and leukemogenesis, therefore, in this work we investigated the epigenetic and metabolic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44 treatment results in an extensive change of histone modifications. We found that inhibiting CBP/p300 (responsible for H3K9ac) and MLL (responsible for H3K4me1/2/3) attenuated the differentiation promoting effects of anti-CD44. Conversely, inhibition of EZH1/2 (responsible for H3K27me3) enhanced anti-CD44’s effects. The observed increase in H3K4me can be caused by an accumulation of succinate, a metabolite that is part of the tricarboxylic acid cycle. Taken together, these data highlight the promising potential of using anti-CD44 either alone or in combination with epigenetic inhibitor as a therapeutic agent across multiple subtypes in AML. Acute myeloid leukemia (AML) is a subset of leukemia characterized by the clonal expansion of abnormal myeloid blasts. The poor prognosis of AML patients caused by low tolerance to intensive chemotherapy and high relapse rates triggered by leukemic stem cells has encouraged the pursuit of alternative therapies. Differentiation therapy, which involves the use of agents that can release the differentiation block in these leukemic stem/blasts has emerged as a promising therapy following the success of All-trans retinoic acid (ATRA) on one AML subtype. Efforts focused on finding differentiation agents that are effective on other AML subtypes brought into light the possibility of using anti-CD44 antibodies to target AML cells. Anti-CD44 has been shown to release the differentiation block in varying AML subtypes and work in our lab is focused on understanding the mechanism by which it induces its effects. Recent studies highlight the role that epigenetic and metabolic regulation plays during hematopoiesis and leukemogenesis, therefore, in this work we investigated the epigenetic and metabolic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44 treatment results in an extensive change of histone modifications. We found that inhibiting CBP/p300 (responsible for H3K9ac) and MLL (responsible for H3K4me1/2/3) attenuated the differentiation promoting effects of anti-CD44. Conversely, inhibition of EZH1/2 (responsible for H3K27me3) enhanced anti-CD44’s effects. The observed increase in H3K4me can be caused by an accumulation of succinate, a metabolite that is part of the tricarboxylic acid cycle. Taken together, these data highlight the promising potential of using anti-CD44 either alone or in combination with epigenetic inhibitor as a therapeutic agent across multiple subtypes in AML.

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.

Newly Developed Oncolytic Herpes Simplex Viruses Expressing Multiple Immunomodulatory Transgenes Effectively Target AML Cells

Acute myeloid leukemia (AML) is the most common human leukemia and is a major area of unmet medical need among hematologic malignancies. Progress has been made in identifying therapeutic targets and several approved therapies, but resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumor specific oncolysis and the generation of a potent adaptive anti-tumor immune response for efficacy. To investigate if our newly developed oncolytic herpes simplex viruses (oHSVs), designed to potentiate anti-leukemia immunity, effectively target primitive AML cells, we evaluated oHSV-VG161, which is engineered to express IL-12, IL-15 and the IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interaction. After screening several AML cell lines that expressed relatively high levels of a HSV entry receptor (HVEM), we demonstrated that VG161-infected OCIAML3 and MOLM13 cells significantly enhanced cell killing (IC 50: 0.4 & 1.8 multiplicity of infection (MOI) as compared to MV4-11 and U973 cells (IC 50: 3.0 & 9.5 MOI). These effects were 2-3 folds lower in control VG160-infected cells. We also observed that VG161-infected AML cells induced apoptosis in a dose-dependent manner (~50%) after 48 hours and cleaved PARP, Caspase-3 and Caspase-8 were increased in these cells, and to a lesser extent in control VG160-infected cells. Both VG160 and VG161 viruses replicated efficiently in OCIAML3 and MOLM13 cells in a timely, dose-dependent manner, evidenced by qPCR detection of HSV-1 ICP27 DNA copy numbers (>500-fold increase) over 48 hours of treatment. This result was supported by detection of protein expression of HSV-1 glycoprotein D in VG160 and VG161-infected cells (up to 40% of protein detected) by FACS analysis. Interestingly, IL-12 but not IL-15 protein expression was found in intracellular-stained VG161-infected OCIAML3 and MOLM13 cells in a dose-dependent manner (up to 13% of protein detected, P<0.01) but not in VG160-infected cells, as assessed by FACS analysis. Production of IL-12 was also detected in cultured media obtained from VG161-infected AML cells (up to 150 pg/mL) by ELISA. To investigate potential molecular mechanisms of VG161-mediated anti-leukemia response and specific signalling pathways, we have screened several potential candidates and found immune regulating genes, such as IRF3, IRF7, IRF9, NFkB and ISGs, as well as type I IFN to be highly increased in VG161-infected cells as compared to VG160-infected cells (2-4-folds, P<0.001) in a dose dependent manner over 48 hours of treatment, assayed by qRT-PCR. Western blot analysis demonstrated increased phosphorylation of p-STAT1 and its protein expression in VG161-infected cells compared to VG160 control cells (~2-fold). These results suggest that VG161 viruses expressing several engineered immunomodulatory transgenes, particularly IL-12, contribute to anti-leukemia responses by activating specific immune regulating pathways such as the JAK/STAT pathway. In addition, we detected an increase in both RNA and protein levels of PD-L1 in VG161-infected AML cells, suggesting the necessity of PD-L1 blocking peptide in the viral construct. To further investigate VG161's role in regulating innate and adaptive immune responses, we have examined the biological effects of VG160/VG161 in the presence of healthy peripheral blood mononuclear cells (PBMC) in both AML cell lines and primary AML patient cells in vitro. Most interestingly, VG160 or VG161-infected OCIAML3 and MOLM13 cells show enhanced cell killing when co-cultured with PBMC and this cell killing effect was greatly enhanced in VG161-infected cells as compared to VG160-infected cells, especially in the MOLM13 cell line (up to 90% killing). This observation was further supported when primitive AML patient cells were co-cultured with VG161 and PBMC as compared to VG160 control cells. Moreover, PD-L1 expression was highly increased in AML patient cells when cultured with VG161 as compared to VG160 (2.7-fold) and this was further enhanced when co-cultured with VG161 and PBMC. Thus, we have demonstrated that newly developed oHSVs engineered with several immunomodulatory transgenes effectively target primitive AML cells, suggesting a potential treatment strategy for AML. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition.A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis.To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells.Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. DisclosuresMinden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

TET3 promotes AML growth and epigenetically regulates glucose metabolism and leukemic stem cell associated pathways.

Acute myeloid leukemia (AML) is considered a poor prognosis malignancy where patients exhibit altered glucose metabolism and stem cell signatures that contribute to AML growth and maintenance. Here, we report that the epigenetic factor, Ten-Eleven Translocation 3 (TET3) dioxygenase is overexpressed in AML patients and functionally validated human leukemic stem cells (LSCs), is required for leukemic growth by virtue of its regulation of glucose metabolism in AML cells. In human AML cells, TET3 maintains 5-hydroxymethylcytosine (5hmC) epigenetic marks and expression of early myeloid progenitor program, critical glucose metabolism and STAT5A signaling pathway genes, which also positively correlate with TET3 expression in AML patients. Consequently, TET3 depletion impedes hexokinase activity and L-Lactate production in AML cells. Conversely, overexpression of TET3 in healthy human hematopoietic stem progenitors (HSPCs) upregulates the expression of glucose metabolism, STAT5A signaling and AML associated genes, and impairs normal HSPC lineage differentiation in vitro. Finally, TET3 depletion renders AML cells highly sensitive to blockage of the TET3 downstream pathways glycolysis and STAT5 signaling via the combination of 2-Deoxy-D-glucose and STAT5 inhibitor which preferentially targets AML cells but spares healthy CD34+ HSPCs.

Targeting acute myeloid leukemia cells by CD33 receptor-specific MoS2-based nanoconjugates

Acute myeloid leukemia (AML) is a highly aggressive type of cancer caused by the uncontrolled proliferation of undifferentiated myeloblasts, affecting the bone marrow and blood. Systemic chemotherapy is considered the primary treatment strategy; unfortunately, healthy cells are also affected to a large extent, leading to severe side effects of this treatment. Targeted drug therapies are becoming increasingly popular in modern medicine, as they bypass normal tissues and cells. Two-dimensional MoS2-based nanomaterials have attracted attention in the biomedical field as promising agents for cancer diagnosis and therapy. Cancer cells typically (over)express distinctive cytoplasmic membrane-anchored or -spanning protein-based structures (e.g., receptors, enzymes) that distinguish them from healthy, non-cancerous cells. Targeting cancer cells via tumor-specific markers using MoS2-based nanocarriers loaded with labels or drugs can significantly improve specificity and reduce side effects of such treatment. SKM-1 is an established AML cell line that has been employed in various bio-research applications. However, to date, it has not been used as the subject of studies on selective cancer targeting by inorganic nanomaterials. Here, we demonstrate an efficient targeting of AML cells using MoS2 nanoflakes prepared by a facile exfoliation route and functionalized with anti-CD33 antibody that binds to CD33 receptors expressed by SKM-1 cells. Microscopic analyses by confocal laser scanning microscopy supplemented by label-free confocal Raman microscopy proved that (anti-CD33)-MoS2 conjugates were present on the cell surface and within SKM-1 cells, presumably having been internalized via CD33-mediated endocytosis. Furthermore, the cellular uptake of SKM-1 specific (anti-CD33)-MoS2 conjugates assessed by flow cytometry analysis was significantly higher compared with the cellular uptake of SKM-1 nonspecific (anti-GPC3)-MoS2 conjugates. Our results indicate the importance of appropriate functionalization of MoS2 nanomaterials by tumor-recognizing elements that significantly increase their specificity and hence suggest the utilization of MoS2-based nanomaterials in the diagnosis and therapy of AML.

Abstract 1048: Targeting BCL-2 and CD38 in models of acute myeloid leukemia reduces tumour burden

Abstract The prognosis for patients with acute myeloid leukemia (AML) remains poor with high mortality rates. It is often not possible to achieve complete remission with current therapy and relapse following treatment is common. New and more targeted treatment approaches are therefore needed to improve outcomes for patients. AML progression and treatment resistance has been associated with overexpression of BCL2. Venetoclax, a BH3 mimetic targeting BCL2, has been shown to effectively target AML cells and induce cell death. It has been approved for the treatment of AML but as with other AML treatments not all patients respond, and others develop treatment resistance. Research has therefore focussed on exploring combination therapies for Venetoclax. Our group has previously shown that mitochondrial transfer from mesenchymal stromal cells (MSC) to AML blasts promotes AML growth and is mediated by CD38. Daratumumab targets CD38 and inhibits this transfer, which results in impaired AML growth and improved animal survival. Here, we investigate the effect of inhibiting both CD38 with daratumumab and BCL2 with Venetoclax on the survival of AML. Primary AML blasts and MSC were isolated from patients' bone marrow in accordance with the Declaration of Helsinki. Flow cytometry was used to measure CD38 and BCL2 expression in AML blasts compared to normal CD34+ progenitor cells. BCL2 expression was significantly higher in AML blasts and CD38 expression was also increased. Cell viability was significantly reduced following treatment with Venetoclax alone, whilst Daratumumab alone or in combination with Venetoclax did not affect AML survival further. However, the action of daratumumab relies on the bone marrow microenvironment and AML blasts were therefore co-cultured with MSC and then treated with Venetoclax or daratumumab or in combination. After 24 hours cells were stained with Annexin V-FITC/PI and flow cytometry was used to assess levels of apoptosis. Combination treatment with Venetoclax and Daratumumab resulted in significantly more apoptosis in AML cells compared to AML cells treated with single agent. Finally, the effect of combination treatment with Venetoclax and Daratumumab was assessed in vivo using an NSG xenograft mouse model of AML. Mice were engrafted with MV411-luc or patient derived AML and then treated with vehicle control (PBS) or daratumumab alone (5mg/kg on day 7 and 14) or Venetoclax alone (100mg/kg/day) or both daratumumab and Venetoclax. Bioluminescence imaging was used to assess disease engraftment and progression before and after treatment. Combining treatment with Daratumumab and Venetoclax in vivo significantly reduced tumour burden and improved animal survival compared to control or single agent. This data supports that combination treatment with Venetoclax and Daratumumab could have an important clinical application in the treatment of AML. Citation Format: Charlotte Hellmich, Jayna J. Mistry, Amelia Lambert, Jamie A. Moore, Aisha Jibril, Angela Collins, Kristian M. Bowles, Stuart A. Rushworth. Targeting BCL-2 and CD38 in models of acute myeloid leukemia reduces tumour burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1048.

Bispecific Anti-CD123 x Anti-CD3 Adaptir™ Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro

Background: The IL-3 receptor CD123 is an attractive target for antibody-based therapies in acute myeloid leukemia (AML) because of its frequent expression on AML blasts and its reported overexpression on leukemic stem cells (LSCs) as compared to normal hematopoietic tissues. Previously, two CD123 x CD3 bispecific targeting ADAPTIR constructs (APVO436 and APVO437) have been shown to bind CD123 with high affinity, engage T-cells and, in the presence of effector T-cells, cause cytotoxicity in CD123-expressing cell lines as well as in an in vivo xenograft model. Here, we further investigated the determinants of anti-tumor efficacy of these constructs in human AML.Materials and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML. CD123 expression on myeloblasts and cells lines and the percentage of endogenous CD3+ T-cells in AML specimens were quantified by flow cytometry. To determine specific cytotoxicity, cell lines or primary AML cells were incubated in culture medium containing various concentrations of ADAPTIR constructs APVO436 and APVO437. Exogenous T-cells isolated from healthy volunteers were labeled with CellVue membrane dye and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; target AML cells were identified by forward/side scatter properties and negativity for CellVue dye. Specific cytotoxicity was calculated as: 100 x (%DAPItreated - %DAPIcontrol)/(100-%DAPIcontrol) and are presented as mean±SEM of all samples tested. T-cell activation, proliferation and depletion of CD123+ cell populations in normal and AML specimens were assessed using multi-color flow cytometry.Results: We examined the ability of APVO436 and APVO437 to induce cytotoxicity in primary human AML specimens. Of 20 samples tested, 10 had both >60% viability on thaw and >40% viability after 48 hours in cultures and were included for further analysis. The median percentage of myeloid blasts and CD3+ T-cells in the analyzed specimens was 78.5% (range 52-91.7%) and 2.95% (range 0.3-8.8%). With no exogenous T-cells, APVO436 and APVO437 induced modest cytotoxicity; at 1000 pM, APVO436 and APVO437 induced specific cytotoxicity of 6.8±3.8% and 3.9±3.6%, respectively. There was no statistically significant correlation between the percentage of endogenous T-cells present in the sample and the amount of cytotoxicity in the absence of exogenous T-cells. The addition of healthy donor T-cells increased cytotoxicity in a E:T-dependent manner (p <0.0001 for both APVO436 and APVO437) and increasing drug concentrations also increased cytotoxicity in a statistically-significant fashion (p <0.0001 for both APVO436 and APVO437). There was no consistent linear relationship between MFICD123 of blasts (MFICD123, arbitrary units) and cytotoxicity. Significant cytotoxicity was achieved even at the lowest levels of blast CD123 expression (MFICD123 range among patient samples: 349-3423). Inclusion of all 20 samples in cytotoxicity analysis did not qualitatively change results. APVO436 induced T-cell activation in both normal and AML samples at 24 hours as assessed by quantification of CD25 and CD69 expressing T-cells. T-cell activation was accompanied by proliferation of T-cells in normal and AML specimens at 96 hours in the presence of APVO436. APVO436 induced dose dependent depletion of CD123+ cells which was evident at 24 hours in both normal and AML samples.Conclusion: APVO436 and APVO437 cause target cell cytolysis in a dose-dependent, target-antigen-dependent, and E:T-dependent manner. Broad activity was seen in primary human AML samples and both constructs were effective in inducing cytotoxicity across a range of blast CD123 expression levels, including specimens with very limited CD123 expression. Endogenous T-cell activation and proliferation accompanied by CD123+ cell depletion demonstrate similar activity of anti-CD123 x anti-CD3 ADAPTIRs in both normal and AML subject samples. These data are supportive of further investigation of anti-CD123 x anti-CD3 ADAPTIR molecules as a potential treatment option for AML. DisclosuresGottschalk:Aptevo Therapeutics: Employment, Equity Ownership. Comeau:Aptevo Therapeutics: Employment, Equity Ownership. Hoyos:Aptevo Therapeutics: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

Antineoplastic effect of a diphtheria toxin-based nanoparticle targeting acute myeloid leukemia cells overexpressing CXCR4

Nanomedicine has opened an opportunity to improve current clinical practice by enhancing the selectivity in the delivery of antitumor drugs to specific cancer cells. These new strategies are able to bypass toxicity on normal cells increasing the effectiveness of current anticancer treatments. In acute myeloid leukemia (AML) current chemotherapy treatments generate a relevant toxic impact in normal cells and severe side effects or even patient death. In this study, we have designed a self-assembling protein nanoparticle, T22-DITOX-H6, which incorporates a ligand (T22) targeting CXCR4-overexpressing (CXCR4+) cells, and a potent cytotoxic diphtheria toxin domain. CXCR4 is overexpressed in AML leukemic cells and associates with poor prognosis, being, therefore, a relevant clinical target. We demonstrate here that T22-DITOX-H6 induces apoptosis in CXCR4+ leukemic cells through CXCR4-dependent internalization. In addition, repeated T22-DITOX-H6 treatment (10 μg/dose per 10 doses, intravenously injected) in a disseminated AML mouse model (NSG mice intravenously injected with THP-1-Luci cells, n = 10 per group) potently blocks the dissemination of AML cells in bone marrow, spleen and liver of treated mice, without inducing toxicity in healthy tissues. In conclusion, our strategy of selectively ablating CXCR4 positive leukemic cells by administering the T22-DITOX-H6 nanoparticle could be a promising treatment, especially in patients undergoing AML relapse after chemotherapy, in which leukemic cells overexpress CXCR4.