AB8939, a Microtubule-Destabilizing Agent with Potential to Overcome Multidrug Resistance, Is Active across the Range (M0-M7) of Acute Myeloid Leukemia Subtypes

Compound AB8939 is a structurally novel, small chemical molecule, synthesized tubulin inhibitor that can circumvent two of the major resistance mechanisms in acute myeloid leukemia (AML), namely P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance, thereby conferring an important advantage over traditional tubulin inhibitors. A series of ex vivo and in vivo studies provide proof-of-concept that AB8939 has broad anti-proliferative activity across the breath of acute myeloid leukemia (AML) subtypes, i.e. M0 through M7 of the French-American-British AML classification. Acute myeloid leukemia blasts were isolated from patients' peripheral blood and/or bone marrow samples, collected either at the time of diagnosis or following relapse, and also from patient derived xenograft (PDX) models. After purification, mononuclear cells were treated for 48 hours with various concentrations of AB8939 or cytarabine (Ara-C) and analyzed in a cell proliferation/viability assay. AB8939 produced a strong anti-proliferative action against blasts isolated from AML patients with a majority of IC50 values ranging from 1.4 nM to 1.0 µM. Two-thirds of AML patients had nanomolar sensitivity to AB8939 (IC50 ≤ 500 nM), while 44% where very sensitive (IC50 ≤ 100 nM) and 11% were highly sensitive (IC50 ≤ 10 nM). The potential of AB8939 to overcome Ara-C-resistance was also evident with 66% of Ara-C-resistant blasts (i.e. IC50 >5 µM) being sensitive to AB8939. Notably, AB8939 demonstrated activity across the entire spectrum of AML subtypes, according to the French-American-British (FAB) AML classification, with an IC50 of < 50 nM in M0, M1, M4, M5 and M6 subtypes, corresponding to over 90% of the AML patient population. A slightly lower sensitivity was observed for the M3 subtype (IC50 = 1.25 µM). Additionally, potent AB8939 activity was also seen in Ara-C-insensitive biphenotypic and mixed-phenotype acute leukemia samples. All FAB categories other than M7 were tested in the abovementioned ex vivo assessment. Acute megakaryocytic leukemia (FAB AML subtype M7) is a rare form of adult AML, accounting for only 1% of cases, and is associated with resistance to standard treatment and poor prognosis. The effect of AB8939 in this subtype was assessed in vivo using an AMKL26 model, an NSG mouse model based on cells isolated from a patient with an aggressive acute megakaryocytic leukemia presenting the ETO2-GLIS2 fusion oncogene. Following post graft detection of blasts, single agent AB8939 was administered intravenously at a dose of 2 mg/kg for three consecutive days per week (3 ON / 4 OFF) for 2 weeks and then at 5 mg/kg for three consecutive days per week (3 ON / 4 OFF) for 1 week. At the end of the 3-week treatment period, blast detection in bone marrow was performed via bioluminescence imaging with comparison to vehicle-treated controls. As seen in the figure below, single agent AB8939 showed strong anti-leukemic activity in this AMKL26 mouse model as evidenced by the near eradication of blasts. No blasts could be detected in 6 out of 8 mice treated with AB8939. At the described dosing schedule, AB8939 was well-tolerated with no toxicity-related deaths and no impact on animal body weight or behavior. These findings provide preclinical proof of concept for the development of AB8939 as a next-generation tubulin inhibitor for AML, in particular for poor-prognosis AML subsets and relapse/refractory AML; i.e. patients that currently have very limited therapeutic options and represent the highest unmet medical need. Disclosures Goubard: AB Science: Employment. Humbert:AB Science: Employment. Mansfield:AB Science: Employment, Patents & Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. AB8939 Study Group:AB Science: Consultancy, Employment.

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A Functional Approach to Precision Medicine Identifies Targeted Therapies for Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.853.853 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 853-853

Author(s):

Shruti Bhatt ◽

Vineeth Kumar Murali ◽

Holly Zhu ◽

Sophia Adamia ◽

Amanda L Christie ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Precision Medicine ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Single Agent ◽

Targeted Agents ◽

Smac Mimetic ◽

Pdx Models ◽

Acute Myeloid

Abstract Identification of genetic heterogeneity in acute myeloid leukemia (AML) has provided a unique opportunity for the greater individualization of therapy. However the implementation of new therapies has lagged far behind the ability to initially recognize operationally important genetic lesions. Until we have further bridged this gap between the identification of genetic lesions and the resultant knowledge of effective therapies, alternative strategies for rapidly identifying candidate therapies can become an important tool for precision medicine. Since most agents, regardless of whether "cytotoxic" or "targeted" ultimately function by activating the mitochondrial apoptotic pathway, we hypothesized that a tool that measures mitochondrial sensitivity may serve as a broadly predictable biomarker. We developed a dynamic BH3 profiling (DBP) technique that measures early death signaling within 8-16 hours after exposure to drugs. Increased cell death signaling is reflected by increased mitochondrial sensitivity (i.e. increased priming) to standardized BH3 peptides mimicking pro-apoptotic proteins. To develop a personalized therapy for AML using DBP, we utilized 20 independent patient derived xenograft (PDX) models, established from de novo, primary refractory or relapsed (R/R) patients (available at http://www.PRoXe.org). Human myeloblasts from spleen and bone marrow of xenotransplanted NSG mice were exposed to 30 targeted and 3 standard of care drugs to determine mitochondrial responses. Unsupervised hierarchical clustering of ex-vivo DBP measurements across AML PDXs revealed several distinct clusters. Majority of targeted agents with an ability to induce priming in selective PDXs were enriched within a cluster, including kinase inhibitors, epigenetic modifiers, SMAC mimetic and chemotherapy drugs. In contrast, a discrete subcluster of drugs showed sensitivity across majority of PDXs, including BH3 mimetics, CDK9 inhibitors and HDAC inhibitors. Drugs with identical mechanism of action showed similar priming patterns across PDXs. Of note, 3 non-myeloid PDXs clustered distinctly from AML, an indication of differential priming responses owing to their cells of origin. AML PDXs developed from treatment naïve patients clustered adjacently and showed greater priming responses to a large number of drugs as opposed to PDXs from R/R patients that formed a discrete cluster. These data reveal that mitochondrial priming can stratify AML PDXs according to its predicted sensitivity to targeted agents. Next, we validated ability of DBP to predict in-vivo responses of single agent birinapant (SMAC mimetic), JQ-1 (BRD-4 inhibitor), quizartinib (FLT-3 inhibitor), and venetoclax (BCL-2 inhibitor) across 6 AML PDX models, prioritized based on their greatest range of priming responses. We found that birinapant was most efficacious nonetheless as expected from ex-vivo DBP studies, responses varied between different PDX models. Myeloblasts of those PDXs that showed the greatest drug-induced changes in apoptotic priming were indeed the PDXs with the highest in-vivo responses. When we compared the ability of DBP to identify sensitive PDXs with additional precision medicine tools such as genomics, we found that DBP was able to accurately predict quizartinib activity in PDXs expressing WT FLT-3, which would have been predicted to be unresponsive based on genomic analysis. Collectively, priming responses obtained from ex-vivo DBP was able to rank different PDX models according to their sensitivities to targeted agents (AUC of ROC curve 0.8731, p<0.005). To investigate if DBP can predict combination therapies in relapsed settings, we first developed resistant models of single agents and then repeated DBP. Myeloblasts from relapsing clone showed reduced overall mitochondrial priming and lacked acquisition of a new chemical dependency compared to initial clone. This suggests that targeting of pre-existing dependencies might be more crucial than therapy induced dependency for AML. In summary, our findings highlight that mitochondria-based measurements could identifying individualized therapy for a heterogeneous population and may serve as a as a powerful biomarker to identify the best responders to patient therapies. Disclosures Letai: AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding.

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APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

Haematologica ◽

10.3324/haematol.2020.259531 ◽

2021 ◽

Author(s):

Rudy Birsen ◽

Clement Larrue ◽

Justine Decroocq ◽

Natacha Johnson ◽

Nathan Guiraud ◽

...

Keyword(s):

Lipid Peroxidation ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Lipid Peroxides ◽

Death Process ◽

Glutathione Biosynthesis ◽

Acute Myeloid

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia. APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

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852 Trifunctional NKp46/CD16a-NK cell engager targeting CD123 overcomes acute myeloid leukemia resistance to ADCC

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.852 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A893-A893

Author(s):

Laurent Gauthier ◽

Angela Virone-Oddos ◽

Jochen Beninga ◽

Benjamin Rossi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Antitumor Activity ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Activating Receptors ◽

Acute Myeloid

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

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A Phase I Study Of Nilotinib As a c-Kit Inhibitor Combined With Re-Induction Chemotherapy For Relapsed and Refractory c-Kit Positive Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3961.3961 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3961-3961

Author(s):

Joseph M. Brandwein ◽

David Hedley ◽

Sue Chow ◽

Karen Yee ◽

Andre C Schuh ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Phase I ◽

Induction Chemotherapy ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Significant Inhibition ◽

Dose Titration ◽

Titration Curves ◽

Acute Myeloid

Abstract Background A previous study in relapsed/refractory AML, combining re-induction chemotherapy with imatinib as a c-kit inhibitor, demonstrated that effective inhibition of downstream pAkt was associated with a higher CR rate (Brandwein et al, 2011). Nilotinib is also a c-kit inhibitor which diffuses into cells and is therefore not affected by potential defects in active cellular influx mechanisms. Methods Patients age 18-65 with acute myeloid leukemia (AML) who had persistent leukemia after 3+7 induction with cytarabine and daunorubicin, or relapsed within 24 months of achieving CR, and whose blasts were c-kit (CD117) positive, were enrolled in a phase I clinical trial with nilotinib on Days 1-12 combined with mitoxantrone 10 mg/m2/day IV Days 6-10, etoposide 100 mg/m2/day IV Days 6-10 and cytarabine 1.5 g/m2 IV q12h x 4 doses on Days 11-12. None of the cases harbored c-kit mutations. Nilotinib was escalated through 2 dose levels (400 mg daily and 400 mg BID) in successive 6 patient cohorts; up to 2/6 dose-limiting toxicities (DLTs) were permitted. Results At the 400 mg once daily nilotinib dose there were 2 DLTs, both hematologic and consisting of prolonged platelet and neutrophil recovery. At the 400 mg BID dose there was one DLT, consisting of grade 3 transient liver enzyme elevation; 5/6 patients experienced transient grade I-II bilirubin elevations at this dose. The median time to ANC > 0.5 x109/L was 38 days (range 28-54) and to platelets > 20 x109/L was 30 days (range 28-54). Complete responses were seen in 10 patients, 5/6 at each dose level. AML blasts from peripheral blood were assayed for pAKT, pERK and pS6 by flow cytometry, after stimulation with stem cell factor (SCF), on Days 1 and 6, pre- and 1.5 and 3 hours post-nilotinib dosing on each day. A decrease in the level of SCF-stimulated pAkt of at least 50% on Day 6 post-nilotinib, as compared to baseline, was seen in 1/6 patients at the 400 mg BID dose; the remaining 11 cases did not demonstrate an appreciable decrease in pAkt and other intermediates as compared to pre-nilotinib levels. Ex vivo dose titration curves showed that nilotinib inhibited c-kit mediated ERK and Akt activation in the blast cells of all cases, but concentrations >10 micromolar were needed to produce 50% inhibition in most cases. Conclusions The results indicate that nilotinib can be safely combined with this re-induction chemotherapy regimen, at doses up to 400 mg BID. Although the CR rate was high, in most cases nilotinib did not demonstrate significant inhibition of the c-kit pathway in leukemia cells in vivo. Dose titration curves showed that concentrations needed to produce 50% inhibition were generally above those achievable in vivo. Disclosures: Brandwein: Novartis: Honoraria, Research Funding. Off Label Use: nilotinib for acute myeloid leukemia.

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Autophagy Promotes the Survival and Therapy Resistance of Human Acute Myeloid Leukemia Cells Under Hypoxia

Blood ◽

10.1182/blood.v124.21.2236.2236 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2236-2236 ◽

Cited By ~ 1

Author(s):

Dirkje W Hanekamp ◽

Megan K Johnson ◽

Scott Portwood ◽

Joshua Acklin ◽

Eunice S. Wang

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase Inhibitor ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Single Agent ◽

Xenograft Models ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring primarily in older adults. Despite high remission rates following upfront therapy, the disease eventually recurs in most patients, and overall cure rates remain only 20-30%. Preclinical studies have recently demonstrated that the marrow microenvironment in acute leukemic hosts to be intrinsically hypoxic, with AML progression associated with further hypoxia. Moreover, human AML cells and primary AML colonies cultured under hypoxia are markedly less sensitive to cytarabine chemotherapy than normoxic cells. We hypothesized that AML cells may respond to hypoxic stress and mediate chemoresistance in part by invoking autophagy, a highly regulated catabolic process by which cells evade apoptosis by degrading damaged cellular components. To test our hypothesis, we investigated the effects of two known autophagy inhibitors (bafilomycin A1 (Baf) and chloroquine (CQ)) on the sensitivity of human AML cells to various therapeutic agents under differing oxygen levels. Methods: We treated HEL (FLT-3 wildtype) and MV4-11 (FLT-3 ITD mutant) AML cells with autophagy inhibitors (Baf and CQ) alone and in combination with a chemotherapeutic drug (cytarabine (AraC), doxorubicin (Dox), decitabine (Dac)) or a tyrosine kinase inhibitor (sorafenib, SFN) under normoxic (21% O2) or hypoxic (1% O2) conditions. Apoptosis /cell death and proliferation were measured by flow cytometry for Annexin-PI and MTT assays, respectively. Autophagy was assessed by flow cytometry using Cyto-ID Green Dye (Enzo Life Sciences), fluorescent microscropy for acridine orange dye accumulation, and western blot analysis. Results: Autophagy in human ALL and AML cell lines was significantly increased following 24-72 hours of hypoxia (1% O2) as compared with normoxia and was a relatively late response to prolonged low oxygen levels (> 24 hours). Treatment with cytotoxic agents (AraC or Dox) or hypomethylating agent (Dac) resulted in a dose-dependent increases in the number of autophagic vesicles in AML cells consistent with autophagy induction. Low-doses of Baf which selectively inhibits the vacuolar H+ ATPase to prevent lysosomal acidification, and CQ, which blocks lysosome-autophagosome fusion by raising the pH of lysosomes and endosomes, both resulted in buildup of autophagic vesicles by flow cytometry consistent with inhibition of autophagic flux in human AML cells. Combination treatment with an autophagy inhibitor (Baf, CQ) and cytotoxic chemotherapy (AraC, Dox) significantly enhanced apoptosis and cell death over single agent therapy. Treatment with Baf combined with hypomethylating therapy (Dac) synergistically improved the anti-leukemic effects as compared with monotherapy (CI 0.09-0.31)(see Figure). The addition of Baf also improved cell death induced by sorafenib (SFN) on FLT-3 ITD mutant human AML cells (MV4;11) (CI 0.36-0.9) (see Figure). Single agent Baf or CQ treatment resulted in significantly higher levels of apoptosis and cell death in AML cells under hypoxia. The anti-tumor activity of almost all combination regimens was consistently improved under hypoxic versus normoxic culture conditions. In vivo CQ treatment (25-50 mg/kg i.p. daily) in preclinical human AML xenograft models significantly inhibited systemic leukemia progression as a single agent. Further experiments investigating the in vivo effects of CQ combined with other chemotherapeutic agents in preclinical AML xenograft models are ongoing. Conclusions: Our data suggest that human AML cells preferentially induce autophagy to promote survival under chronic hypoxia and following cytotoxic, hypomethylating, and FLT-3 tyrosine kinase inhibitor therapy. Strategies targeting autophagy therefore may have the potential to improve therapeutic responses and overcome chemoresistance of AML cells within the hypoxic bone marrow microenvironment. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.3302.3302 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3302-3302

Author(s):

Timothy Pardee ◽

Evan Gomes ◽

Jamie Jennings-Gee ◽

David L. Caudell ◽

William Gmeiner

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Single Agent ◽

The Novel ◽

Highly Active ◽

Ic50 Values ◽

Acute Myeloid

Abstract Abstract 3302 Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles. The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC50 values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC50 values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC50 values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM). We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10]. We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation. A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU. In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin. Disclosures: Gmeiner: Salzburg Therapeutics: Equity Ownership.

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Development and characterization of a cyclophosphamide-resistant subline of acute myeloid leukemia in the Lewis x Brown Norway hybrid rat

Blood ◽

10.1182/blood.v76.6.1209.bloodjournal7661209 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1209-1213

Author(s):

TM Koelling ◽

AM Yeager ◽

J Hilton ◽

DT Haynie ◽

JM Wiley

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Alkylating Agents ◽

Leukemic Cells ◽

Brown Norway ◽

Lewis X ◽

Similar Dose ◽

Acute Myeloid

Preclinical studies of resistance to alkylating agents in the Lewis x Brown Norway hybrid (LBN) rat model of acute myeloid leukemia (AML) have hitherto been limited by the sensitivity of LBN AML cells to cyclophosphamide (CY). We developed a CY-resistant subline of LBN AML by serial intravenous (IV) passage of AML cells followed by in vivo exposure to CY (100 mg/kg) 14 days later. After 18 and subsequent passages, CY-treated AML cells remained viable despite ex vivo incubation with 70 to 100 mumol/L 4-hydroperoxycyclophosphamide (4HC) or in vivo exposure to 100 to 300 mg/kg of CY. Once established, resistance to incubation with 4HC was stable in LBN AML cells after at least six serial in vivo passages without exposure to CY. Nevertheless, both control and CY-treated AML cells demonstrated similar dose- dependent sensitivity to 100 to 500 mumol/L phosphoramide mustard (PhM), the active alkylating end-product of CY activation in vivo. Levels of aldehyde dehydrogenase (ALDH), which inactivates CY by prevention of formation of PhM, were significantly elevated in these CY- resistant AML cells: cytosolic and particulate ALDH fractions from these cells were 11 to 13 times control with NAD cofactor and propanal substrate and three to four times control with NADP cofactor and benzaldehyde substrate. Further studies with this animal model of AML, in which resistance to CY is mediated by elevated ALDH activity, may elucidate mechanisms for effective elimination of drug-resistant leukemic cells ex vivo and in vivo.

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Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia

Blood ◽

10.1182/blood.2021012629 ◽

2021 ◽

Author(s):

Jan-Erik Messling ◽

Karl Agger ◽

Kasper L Andersen ◽

Kristina Kromer ◽

Hanna Maria Kuepper ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Synthesis ◽

Myeloid Leukemia ◽

Leukemic Cell ◽

Leukemic Cells ◽

Proof Of Concept ◽

Molecule Inhibitor ◽

Novel Target ◽

Acute Myeloid

Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed as current treatments do not cure the majority of AML patients. Here, we report on a domain-focused, kinome-wide CRISPR-Cas9 screen to identify protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. We show that loss of RIOK2 leads to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, we demonstrate that the ATPase function of RIOK2 is required for cell survival. By using a small molecule inhibitor, we show that pharmacological inhibition of RIOK2 similarly leads to loss of protein synthesis and apoptosis and affects leukemic cell growth in vivo. Our results provide proof-of-concept for targeting RIOK2 as a potential treatment for AML patients.

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Pre-Clinical Activity of Amino-Alcohol Dimeric Naphthoquinones as Potential Therapeutics for Acute Myeloid Leukemia

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520621666210602131558 ◽

2021 ◽

Vol 21 ◽

Author(s):

Dana Ferraris ◽

Rena Lapidus ◽

Phuc Truong ◽

Dominique Bollino ◽

Brandon Carter-Cooper ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Amino Alcohol ◽

Myeloid Leukemia ◽

Single Agent ◽

Xenograft Model ◽

Clinical Activity ◽

Lead Compound ◽

Acute Myeloid

Background: The clinical outcomes of patients with acute myeloid leukemia (AML) remain unsatisfactory, therefore the development of more efficacious and better-tolerated therapy for AML is critical. We have previously reported the anti-leukemic activity of synthetic halohydroxyl dimeric naphthoquinones (BiQ) and aziridinyl BiQ. Objective: This study aimed to improve the potency and bioavailability of BiQ compounds and investigate the anti-leukemic activity of the lead compound in vitro and in a human AML xenograft mouse model. Methods: We designed, synthesized, and performed structure-activity relationship of several rationally designed BiQ analogues that possess amino alcohol functional groups on the naphthoquinone core rings. The compounds were screened for anti-leukemic activity and the mechanism as well as in vivo tolerability and efficacy of our lead compound was investigated. Results: We report that a dimeric naphthoquinone (designated BaltBiQ) demonstrated potent nanomolar anti-leukemic activity in AML cell lines. BaltBiQ treatment resulted in the generation of reactive oxygen species, induction of DNA damage, and inhibition of indoleamine dioxygenase 1. Although BaltBiQ was tolerated well in vivo, it did not significantly improve survival as a single agent, but in combination with the specific Bcl-2 inhibitor, Venetoclax, tumor growth was significantly inhibited compared to untreated mice. Conclusion: We synthesized a novel amino alcohol dimeric naphthoquinone, investigated its main mechanisms of action, reported its in vitro anti-AML cytotoxic activity, and showed its in vivo promising activity combined with a clinically available Bcl-2 inhibitor in a patient-derived xenograft model of AML.

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Mass Cytometry Analysis Of Myelofibrosis and Secondary Acute Myeloid Leukemia Reveals Constitutive and Cytokine Induced Signaling Abnormalities With Differential Sensitivities To Ruxolitinib

Blood ◽

10.1182/blood.v122.21.1610.1610 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1610-1610

Author(s):

Daniel A.C. Fisher ◽

Gregory K. Behbehani ◽

Garry P. Nolan ◽

Sean C. Bendall ◽

Stephen T. Oh

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Jak2 V617f ◽

Signaling Molecules ◽

Mass Cytometry ◽

Secondary Acute Myeloid Leukemia ◽

Acute Myeloid

Abstract The classic myeloproliferative neoplasms (MPNs), including primary and secondary myelofibrosis (MF), are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations induce hyperactivated JAK-STAT signaling in cell lines and mouse models. Other mutations have been found recurrently in human MPNs, however, including mutations associated with transformation of chronic MPNs to secondary acute myeloid leukemia (sAML). The aggregate effects of these mutations on in vivo myeloproliferative signaling and disease phenotypes are not yet well understood. While targeted inhibitors of JAK2 have shown activity in MPNs, evidence of a selective effect on the underlying malignant clone has been lacking. These findings suggest that dysregulation of other signaling molecules may be important in MPN pathogenesis, and that therapeutic targeting of these molecules could be beneficial. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed. Mass cytometry is a novel technology that merges flow cytometry with mass spectrometry and enables the simultaneous measurement of 30+ parameters at the single cell level. We utilized this approach to examine bone marrow or peripheral blood samples from four MF patients and five sAML patients, as well as three normal controls. Cells were exposed to five different perturbation conditions ex vivo, including the cytokines thrombopoietin (TPO) and G-CSF, and/or the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 18 surface markers and 16 intracellular signaling effectors and analyzed on a CyTOF mass cytometer. Single cell data was analyzed using traditional gating strategies, as well as with SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets based on shared surface marker expression. These methods grouped cells into distinct cell populations including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict basal and induced activation of each intracellular marker. MF and sAML patient samples exhibited abnormal cytokine-induced signaling that varied in a patient-specific manner. HSPC responses to TPO ranged from hyposensitive to hypersensitive compared with normal. Markedly abnormal basal (unstimulated) signaling in the HSPC compartment was observed in all MF and sAML patients. Each sample exhibited increased total IkBα and/or pCREB, typically accompanied by elevated phosphorylation of one or more additional signaling molecules. Both basal elevation and cytokine hypersensitivity were frequently observed within a MAP kinase signaling axis represented by pERK and downstream targets pCREB and pS6. Conversely, repressed basal STAT1 phosphorylation was observed in all patients. These abnormalities were not exclusive to sAML versus MF, nor specific to patients with or without the JAK2 V617F mutation. Widespread dysregulation of total IkBα, pERK-pCREB-pS6, and pSTAT1 suggest that these signaling effectors may be characteristic of a general myeloproliferative signaling phenotype. Ex vivo ruxolitinib effectively inhibited the majority of observed hypersensitive cytokine-stimulated signaling effects. In contrast, most basal signaling elevations were insensitive to ruxolitinib. The signaling molecules most frequently insensitive to ruxolitinib were those most frequently basally elevated in MF and sAML HSPC, namely pCREB and total IkBα. In contrast, phosphorylation of the JAK2 substrates STAT3 and STAT5 was almost always sensitive to ruxolitinib. These results suggest that targeting of myeloproliferative signaling pathways either downstream or independent of JAK2 activity may be valuable for improved treatment of MF and sAML patients. Ongoing experiments are focused on determining whether constitutive ruxolitinib-insensitive signaling abnormalities can be identified in patients treated with ruxolitinib in vivo, which may underlie the incomplete responses observed clinically. Taken together, these approaches will provide a deeper understanding of altered signaling networks in the context of targeted therapies in MPNs. Disclosures: Oh: Incyte: Consultancy, Research Funding, Speakers Bureau.

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