Abstract: Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075). Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance. Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27. We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009). Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining "stemness" of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced "stemness" of AML cells (Figure 1c). Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress. References: 1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992. 2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4. Figure 1 Figure 1. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Andreeff: Medicxi: Consultancy; Syndax: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Oxford Biomedica UK: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees. Borthakur: University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; GSK: Consultancy.

Abstract: BACKGROUND: Acute myelogenous leukemia (AML) cells that reside in bone marrow (BM) receive a great deal of protection from the cytotoxic effects of therapeutic agents. In contrast, the circulating leukemia cells are typically more chemosensitive compared to those embedded in BM niches. The BM homing of AML cells is mediated by multiple adhesive and chemokinetic interactions including, respectively, by sialylated glycoproteins on the cancer cells binding to E-selectin on the endothelium and by CXCR4-mediated sensing of SDF-1/CXCL12 gradients. Consequently, both E-selectin and CXCR4 inhibitors have been pursued as targets for add-on therapeutic strategies aimed to "mobilize" AML cells out of the BM. A next generation "mobilizing" agent GMI-1359 combines, in one molecular structure, the E-selectin and CXCR4 inhibitory moieties. In a previous study, GMI-1359 markedly reduced leukemia cell adhesion to endothelial cells, leukemia cellularity in BM (Zhang et al., 2016) and it enhanced the survival of cytarabine/daunorubicin therapy in a FLT3-mutated AML model (Zhang et al., 2015). APPROACH: Here, we used intravital 2-photon microscopy of calvarial BM to study the behavioral response of AML cells to the dual CXCR4 and E-selectin inhibition (GMI-1359) in vivo. We used a mTurquoise2 fluorescence-tagged transplantable mouse AML model (characterized by MLL, ENL-FLT3, ITD, p53-/-). To delineate the bone and vascular niches, the syngeneic immune-competent recipient mice harbored cell lineage fluorescence reporter genes such as Col2.3-GFP, hCD2-DsRed and CD11c-EYFP with the bone collagen and blood highlighted by, respectively, second harmonic generation (SHG) and fluorescent dextran. Intravenously infused AML cells (5x10E4) homed to BM where they gradually displaced most endogenous cells. In this experimental system, GMI-1359 or vehicle were infused into the tail vein while recording AML cell motilities in calvarial BM stroma in 3-D over 4 hours. RESULTS: In untreated or control vehicle treated mice, AML cells were slowly motile, migrating with the average velocity of ~2 µm/min. Cellular trajectories were random within the average 400 µm confinement radius corresponding to the size of BM cavities. Most AML cells localized in the vascular niche, defined as within a 50 µm distance to the nearest blood vessel. A minority of AML cells were also present in the proximity to osteoblasts and the bone (i.e. in the bone niche). In contrast, GMI-1359 infusion resulted in a 66% increase of the average speed of cellular motility of AML cells within 20 min and up to 100% increase within 3.5 hours. The motility pattern remained largely random within the bone cavity confinement volume and was concentrated in the vicinity of vasculature. Thereafter, we observed multiple instances of AML cell intravasation into the capillary lumens followed by several minute-long vessel wall attachment and sudden outflow. This cellular dynamics resulted in a substantial, yet structurally biased decrease of BM AML cellularity whereby the vascular niche was emptied preferentially whilst the bone niche remained populated by AML cells. In no case we observed a complete BM depletion of AML cells. Interestingly, as we reported before, a single, CXCR4-only inhibitor caused a protracted (24-48 h) depletion of AML cells in BM without significant cellular motility enhancement. CONCLUSIONS: Our observations reveal an unexpected mechanism of action by a dual E-selectin/CXCR4 inhibitor involving cellular migratory motility enhancement in BM stroma prior to AML cell intravasation. We envisage that, besides the designed blocking of E-selectin and CXCR4 molecules from binding their corresponding ligands, the dual moiety GMI-1359 agent has a capacity to generate motility-enhancing signals in AML cells that single inhibitors do not seem to trigger. Among several mechanisms possible, this action could involve E-selectin and CXCR4 crosslinking by a dual moiety molecular structure or binding avidity enhancement, requiring further investigation. In addition, our results highlight a likely mechanism of AML resistance to therapeutic "mobilization", one that involves cancer cell persistence in the bone/osteoblastic niches. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Abstract: Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

Abstract: BH3 mimetics are used as an efficient strategy to induce cell death in several blood malignancies, including acute myeloid leukemia (AML). Venetoclax, a potent BCL-2 antagonist, is used clinically in combination with hypomethylating agents for the treatment of AML. Moreover, MCL1 or dual BCL-2/BCL-xL antagonists are under investigation. Yet, resistance to single or combinatorial BH3-mimetic therapies eventually ensues. Integration of multiple genome-wide CRISPR/Cas9 screens revealed that loss of mitophagy modulators sensitizes AML cells to various BH3 mimetics targeting different BCL-2 family members. One such regulator is MFN2, whose protein levels positively correlate with drug resistance in patients with AML. MFN2 overexpression is sufficient to drive resistance to BH3 mimetics in AML. Insensitivity to BH3 mimetics is accompanied by enhanced mitochondria–endoplasmic reticulum interactions and augmented mitophagy flux, which acts as a prosurvival mechanism to eliminate mitochondrial damage. Genetic or pharmacologic MFN2 targeting synergizes with BH3 mimetics by impairing mitochondrial clearance and enhancing apoptosis in AML. Significance: AML remains one of the most difficult-to-treat blood cancers. BH3 mimetics represent a promising therapeutic approach to eliminate AML blasts by activating the apoptotic pathway. Enhanced mitochondrial clearance drives resistance to BH3 mimetics and predicts poor prognosis. Reverting excessive mitophagy can halt BH3-mimetic resistance in AML. This article is highlighted in the In This Issue feature, p. 1501

Abstract: Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA). Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p & lt;0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively). In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts. Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. Figure 1 Figure 1. Disclosures Fogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

Abstract: INTRODUCTION: CXCR4 chemokine receptor inhibitors such as BL-8040 (BioLineRx) have been investigated by us and others as possible anti-leukemic drugs due to their ability to "mobilize" leukemia cells out of the BM and into the circulation, where they are more sensitive to chemotherapy. However, the exact mode of cell relocation remains unclear. CXCR4/CXCL12 signaling pathway also participates in BM homing of immune cells, including both central memory T cells and immunosuppressive CD4+FoxP3+ T-regulatory cells (T-reg). Therefore, CXCR4 inhibition has the potential to either counteract or enhance the process of AML immune surveillance. Therefore, we sought to develop a syngeneic AML model for intravital 2-photon microscopy (TPM) compatible with existing immune reporter mouse strains, which typically occupy the green, yellow and red fluorescence channels. HYPOTHESIS: CXCR4 inhibition decreases AML and T cell BM cellularity by increasing the rate of intravascular cell entry and/or decreasing the rate of circulating cell homing back to BM. MODEL: The cyan-colored fluorescent protein mTurquoise2 was lentivirally introduced into C57BL6-origin AML cells containing the MLL, ENL-FLT3, ITD, and p53-/- mutations, termed AML1-mTurq2. Syngeneic FoxP3-GFP/CD11c-YFP/hCD2-DsRed reporter mice were generated by inter-breeding of the corresponding strains, respectively highlighting T-reg, myeloid antigen presenting cells, and all T cells. After intravenous infusion of 1E5 AML1-mTurq2 cells, 1-2% blasts appeared in peripheral blood on day 9, increasing to 70% on day 15-20 when animals had to be euthanized. TREATMENT: Mice with 〉 1% blasts were given BL-8040 I.P. in two daily 400 µg doses followed by imaging 24 h later, or intravenously during imaging 10 µg and 50 µg one hour later. ANALYSIS: Disease progression was characterized by blood flow cytometry, symptom scoring and thick-mount organ tissue fluorescence microscopy. Intravital TPM of the calvarial bone marrow (BM) was performed through intact bone under general anesthesia. By interline multiplexing dual femtosecond lasers with four-sensor detection for 8 distinct channels, mTurquoise2 and SHG were recorded by the same sensor at, respectively, 860 and 990 nm excitation, along with GFP, YFP, DsRed and dextran-TRITC (blood tracer). AML and T cell subsets were 3-D tracked using Imaris software. RESULTS: AML1-mTurq2 cells stably and uniformly expressed bright cyan fluorescence, suitable for intravital TPM with low incident laser powers and fast imaging rates in deep tissue locations. In C57BL6 mice, sparse AML cell clusters were found in BM perivascular spaces on day 1 after cell infusion. AML cells were slowly motile (~4 um/min) and highly proliferative, gradually filling BM spaces and emerging in other organs. T cells and CD11c dendritic cells were present in leukemic BM, and the vasculature appeared largely intact and well perfused. T cells interacted with AML cells and the stroma, migrating with high average velocities (~10 µm/min) and slowing down to ~3 µm/min in late-stage disease. After 2 days of BL-8040 treatment, disease symptom scores improved from 3 to 1 while the untreated controls progressed from 3 to 4 (range 0-6). TPM revealed a 4-fold reduction of AML cellularity in BM. Cellular velocities of both AML and T cells were unchanged by BL-8040 treatment. After acute drug administration, a fraction of stromal AML cells begun entering capillary vessel lumens by amoeboid movement. The intravasated AML cells adhered to vessel wall for 1-2 minutes before rapid detachment. Some cells remained tethered while already loose in the blood stream. CONCLUSIONS: A novel, brightly cyan-fluorescent syngeneic AML1-mTurq2 AML model is advantageous for 6-color intravital microscopy of cell trafficking and immune surveillance in optimal compatibility with green, yellow and red reporters of cell lineages and tissue architecture. Using this model, we show that CXCR4 inhibitor BL-8040 decreases AML BM cellularity by increasing the frequency of intravasation without increasing AML migratory velocity. Disclosures Zal: Daiichi-Sankyo: Research Funding; NIH-CTEP: Research Funding; BioLineRx: Research Funding; VueBio.com: Equity Ownership; NIH/NCI: Research Funding; CPRIT: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy.

Abstract: Lymphocytes encounter varying oxygen levels as they traverse through healthy and diseased tissue. Hypoxia is a hallmark of the tumor microenvironment and can affect the anti-tumor immune response. However, despite the importance of T cell adaptation to differing oxygen concentrations in the tumor niche, there has been no method to study T cell spatiotemporal dynamics in the context of oxygen in vivo. To this end, we developed phosphorescence-optimized 2-photon lifetime and kinetic (2pOLAK) microscopy which enables co-imaging of phosphorescence lifetimes and cellular dynamics in highly fluorescent biological reporter systems. In conjunction with the PtP-C343 oxygen probe, 2pOLAK microscopy revealed the tissue oxygen landscapes and individual oxygen “experiences” of T cells as they moved through tissues in syngeneic models of metastatic lung cancer and acute leukemia. We found that T cells experienced hypoxia in leukemic bone marrow, and that the motility of these cells was significantly decreased relative to non-hypoxic T-cells in healthy bone marrow. Inhibition of oxidative phosphorylation slowed non-hypoxic T cell motility to a level comparable with that of hypoxic T cells. T cell motility was also decreased in hypoxic lung tumor cores, and it was significantly lower than in the tumor margin, where T cells experienced higher oxygen. Supplemental oxygenation increased the oxygen experienced by T cells in the tumor core and reanimated T cell motility. These studies describe a novel method for co-imaging tissue oxygen and cellular behavior, shed light on the role that oxygen availability plays in T cell dynamics in vivo, and suggest that counteracting hypoxia can improve tumor immune surveillance by restarting T cell motility.

Abstract: Background: Acute myeloid leukemia (AML) is a heterologous hematological malignancy in which the p53-mutated subset is associated with the most guarded prognosis. Induction therapy for AML with cytarabine (cytosine arabinoside, ARA-C) is routinely used in combinations with anthracyclines. Annamycin (ANN) is an antitumoral anthracycline whose anti-leukemia activity, in contrast to doxorubicin (DOX) and daunorubicin, is unaffected by P-glycoprotein (ABCB1)-related multidrug resistance (MDR1). Unlike conventional anthracyclines, ANN accumulates in multidrug resistant cell lines, inducing DNA damage and apoptosis. Additionally, in preclinical toxicology studies, ANN displayed a greatly reduced cardiotoxicity profile compared to DOX. A liposomal formulation of ANN, termed L-Annamycin (L-ANN), is being evaluated in patients with acute myeloid leukemia (AML) in two phase Ib/IIa clinical trials in both the US and Europe. The patients are stringently followed for cardiotoxicity (Shephard et al., ASH 2020, submitted). There was no decrease in ejection fractions observed in any of the 20 patients treated to date and all cardiotoxicity biomarkers including troponin remained normal. In addition, echocardiogram analyses by the Duke cardio-oncology lab, and independently by a cardio-oncologist at Cleveland Clinic, were all normal. Considering that cardiotoxicity and MDR1-limited anthracyclines like daunorubicin are routinely used in a combination with ARA-C as induction therapy in AML, we here evaluated the combination of Ara-C with L-ANN pre-clinically. Objective: The objective of the study was to assess in vivo efficacy of the combination of L-ANN with ARA-C in a pre-clinical model of AML. Methods: The efficacy of L-ANN alone or in combination with ARA-C was investigated in a highly aggressive AML mouse model characterized by the p53-/-, MLL, ENL-FLT3, ITD mutations and genetically tagged with the cyan fluorescent protein mTurquoise2 for flow cytometry and microscopic visualization. L-ANN was intravenously administered at different dosing regimens (days 1, 2, 3 or 1, 3, 5 weekly, or 1 dose of 4 mg/kg once a week). ARA-C was administered by 5 daily intraperitoneal injections at 50 mg/kg, which was repeated every other week up to 3 times. The level of leukemia cells in peripheral circulation was analyzed by flow cytometry and AML cell presence in organ tissues was imaged by thick-mount fresh tissue confocal microscopy, in various disease stages. Results: In all tested L-ANN administration regimens (days 1, 2, 3 or 1, 3, 5 weekly, or 1 dose of 4 mg/kg once a week), we observed a significant increase in the survival of ANNARAC cohorts (combination of L-ANN with ARA-C), when compared with the respective single agents. Specifically, upon intravenous infusion of 1x105 AML1-mTurq2 cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease developed with median survival of 14 days. Administration of L-ANN on a weekly basis significantly delayed leukemia progression, as evaluated by flow cytometry and fluorescence microscopy, resulting in survival increase to 34 to 40 days, in multiple experiments. The mice treated with 50 mg/kg of ARA-C daily for 5 days a week every other week using intraperitoneal injections showed moderate to limited response to the therapy with median survival ranging from 17 to 30 days. In contrast, the median survival of animals treated with the L-ANN/ARA-C combination using different schedules ranged from 44 to 76 days, with a fraction of animals living more than 180 days after implantation of AML cells. Remarkably, imaged on day 36, the bone marrow, spleen and lungs of mice receiving combination of L-ANN (4 mg/kg once a week) with ARA-C (50 mg/kg five times per week) showed no residual disease. These results are consistent with the increased survival observed for this combination. Conclusion: This study demonstrated vastly higher efficacy of the L-ANN/ARA-C combination (ANNARAC) over that of the single agents in an immune-competent setting of an aggressive, p53-null AML model. Overall, these experiments indicate that L-ANN has the capacity to sensitize AML cells to the ARA-C induction regimen and support initiation of clinical development of L-ANN in combination with ARA-C in AML patients. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Zielinski:CNS Pharmaceuticals: Current equity holder in private company, Patents & Royalties; Moleculin Biotech, Inc.: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding. Grela:Moleculin Biotech, Inc.: Current Employment, Current equity holder in private company, Patents & Royalties. Skora:Moleculin Biotech, Inc.: Current equity holder in private company, Patents & Royalties; CNS Pharmaceuticals: Current equity holder in private company, Patents & Royalties. Fokt:CNS Pharmaceuticals: Current equity holder in private company, Patents & Royalties; Moleculin Biotech, Inc.: Consultancy, Current equity holder in private company, Patents & Royalties, Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding. Shephard:Moleculin Biotech, Inc.: Current Employment, Current equity holder in private company, Patents & Royalties. Priebe:Animal Life Sciences: Current equity holder in private company, Other: Scientific Advisor; WPD Pharmaceuticals: Current equity holder in publicly-traded company, Other: Chairman of Scientific Advisory Board, Patents & Royalties, Research Funding; CNS Pharmaceuticals: Current equity holder in private company, Other: Chairman of Scientific Advisory Board, Patents & Royalties, Research Funding; Moleculin Biotech: Current equity holder in publicly-traded company, Other: Membership of Scientific Advisory Board, Patents & Royalties, Research Funding; Reata Pharmaceuticals: Current equity holder in publicly-traded company; Houston Pharmaceuticals: Current equity holder in private company.