WO2021094827A1

WO2021094827A1 - Use of bisantrene to treat measurable residual disease in acute myeloid leukemia

Info

Publication number: WO2021094827A1
Application number: PCT/IB2020/000939
Authority: WO
Inventors: Daniel Tillett
Original assignee: Race Oncology Ltd.
Priority date: 2019-11-11
Filing date: 2020-11-11
Publication date: 2021-05-20

Abstract

The present invention provides a new paradigm for treating of acute myeloid leukemia (AML) focusing on the elimination of measurable residual disease (MRD) by the administration of bisantrene, an antineoplastic agent that has multiple mechanisms of action, including DNA intercalation, inhibition of topoisomerase, and activation of the immune system. The bisantrene can be administered with other antineoplastic agents for the treatment of AML and can be followed by hematopoietic stem cell transplantation. The present invention also is directed to pharmaceutical compositions formulated for the treatment of MRD. The methods and pharmaceutical compositions according to the present invention can employ a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and β-cyclodextrin.

Description

USE OF BISANTRENE TO TREAT MEASURABLE RESIDUAL DISEASE IN ACUTE

MYELOID LEUKEMIA

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application Serial No. 62/933,802 by Dr. Daniel Tillett, entitled “Use of Bisantrene to Treat Measurable Residual Disease in Acute Myeloid Leukemia,” and filed on November 11 , 2019, the contents of which are hereby incorporated in their entirety by this reference.

FIELD OF THE INVENTION

[0002] This invention is directed to the use of bisantrene to treat measurable residual disease in acute myeloid leukemia, either alone or in combination with other drugs, as well as diagnostic methods for the detection of measurable residual disease and the suitability of the use of bisantrene in patients with such disease.

BACKGROUND OF THE INVENTION

[0003] Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cells. Symptoms may include feeling tired, shortness of breath, easy bruising and bleeding, and increased risk of infection. Occasionally, spread may occur to the brain, skin, or gums. As an acute form of leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated. [0004] Risk factors for AML include smoking, previous chemotherapy or radiation therapy, myelodysplastic syndrome, and exposure to the chemical benzene or certain other organic compounds, particularly aromatic compounds. The underlying mechanism involves replacement of normal bone marrow with leukemic cells, which results in a drop in erythrocytes, platelets, and normal leukocytes.

[0005] In 2015, AML affected about one million people and resulted in 147,000 deaths globally. It most commonly occurs in older adults. Males are affected more often than females. AML is curable in about 35% of people under 60 years old and 10% over 60 years old. Older people whose health is too poor for intensive chemotherapy have a typical survival of 5-10 months. It accounts for roughly 1.8% of cancer deaths in the United States.

[0006] Conventional therapy for AML involves induction chemotherapy with the nucleotide analog cytarabine and an anthracycline such as daunorubicin or idarubicin; other drugs can also be used, such as gemtuzumab ozogamicin. The standard combination is the 7+3, with a 7-day continuous infusion of cytarabine at the dosage of 100 or 200 mg/m² per day on days 1 to 7 and daunorubicin at 60 mg/m² per day on days 1 to 3 (H. Dombret & C. Gardin, “An Update of Current Treatments for Adult Acute Myeloid Leukemia,” Blood 127: 53-61 (2015)). Subsequent treatment following induction chemotherapy typically includes hematopoietic stem cell transplantation (HSCT).

[0007] About 60-80% of AML patients go into remission (CR) after induction chemotherapy as described above. However, in such patients, subsequent successful HSCT depends largely on the patient’s measurable residual disease (MRD) status. In MRD(+) patients, the two-year survival rate is less than 25%. In MRD(-) patients, the survival rate is 80%. [0008] Measurable residual disease (MRD) (previously typically referred to as minimal residual disease), is an independent, post-diagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk stratification and treatment planning in conjunction with other well-established clinical, cytogenetic, and molecular data. Traditionally, the threshold for MRD was a morphology-based threshold of 5% blasts. However, current measurement technology has defined the threshold for MRD as down to levels of 1 : 10⁴ to 1 : 10⁶ white blood cells, with the exact threshold dependent on the particular measurement technology employed.

[0009] As stated above, different platforms are available for detecting MRD.

Two methods are currently applied: multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (qPCR), and other methods are currently emerging, including digital PCR and next-generation sequencing (NGS).

[0010] For flow-cytometric MFC MRD assessment, the general approach is to detect the presence of leukemia-associated immunophenotypes (LAIPs) and assess the difference from normal. This can include, but is not necessarily limited to, the detection of early markers such as CD34 and CD117, myeloid-lineage associated markers, and differentiation antigens such as CD2, CD7, CD19, or CD56, in order to track aberrant AML blast cells.

[0011] Alternatively, there are two general approaches for molecular MRD assessment: real-time PCR-based approaches and sequencing approaches wherein sequences from individual DNA/complementary DNA (cDNA) molecules are generated. The PCR approach includes classical real-time qPCR using fluorescent probes, digital PCR, and molecular chimerism analysis. This approach is usually of high sensitivity and therefore currently considered the gold standard. However, its applicability is limited to the ~40% of AML patients that harbor one or more suitable abnormalities.

NGS for MRD assessment can be, at least theoretically, applied to all leukemia-specific genetic aberrations. Markers for molecular assessment can include, for example, the persistent presence of NPM1 mutations and the fusion genes RUNX1-RUNX1T1 , CBFB-MYH11 , and PML-RARA, which can be strong predictors of relapse. In general, the presence of MRD is clearly associated with increased relapse risk and shorter survival for AML (G.J. Schuurhuis et al. , “Minimal/Measurable Residual Disease in AML: A Consensus Document from the European LeukemiaNet MRD Working Party,” Blood 131: 1275-1291 (2018); S.D. Freeman et al., “Prognostic Relevance of Treatment Response Measured by Flow Cytometric Residual Disease Detection in Older Patients with Acute Myeloid Leukemia.” J. Clin. Oncol. 31: 4123-4131 (2013)).

[0012] At present, there are no approved treatments that can change MRD status for AML patients and thus improve transplant outcome. Therefore, there is a need for such treatments that change MRD status for AML patients and thus can improve transplant outcome.

SUMMARY OF THE INVENTION

[0013] The present invention provides a new paradigm for treating of acute myeloid leukemia (AML) focusing on the elimination of measurable residual disease (MRD) by the administration of bisantrene, an antineoplastic agent that has multiple mechanisms of action, including DNA intercalation, inhibition of topoisomerase, and activation of the immune system. This meets the needs for treatments that change MRD status for AML patients and thus can improve transplant outcome.

[0014] One aspect of the present invention is a method of converting an AML patient, subsequent to initial treatment, from MRD(+) status to MRD(-) status by administering a therapeutically effective quantity of bisantrene to the patient.

[0015] Typically, the bisantrene is administered for 7 days at a dosage from about 200 mg/m²/day to about 300 mg/m²/day. Preferably, the bisantrene is administered for 7 days at a dosage from about 225 mg/m²/day to about 275 mg/m²/day. More preferably, the bisantrene is administered for 7 days at a dosage of about 250 mg/m²/day. Alternatively, the bisantrene is administered for a period of 5 days, 6 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0016] Typically, the bisantrene is administered intravenously, either centrally or peripherally.

[0017] The bisantrene can be administered either as a drug compound or as a pharmaceutical composition, wherein the pharmaceutical composition includes at least one pharmaceutically acceptable carrier. Alternatively, the bisantrene can be administered in a liposome.

[0018] In yet another alternative, the bisantrene is administered in a formulation comprising a bioavailability enhancer selected from the group consisting of mPEG-b- PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b-cyclodextrin. In this alternative, the dosage of bisantrene can be about 5 mg/kg once every other day, 10 times.

[0019] The method can further comprise administering at least one additional therapeutic agent for the treatment of AML.

[0020] The at least one additional therapeutic agent for the treatment of AML can be selected from the group consisting of:

(1) arsenic trioxide;

(2) daunorubicin hydrochloride;

(3) cyclophosphamide;

(4) cytarabine;

(5) glasdegib maleate;

(6) dexamethasone;

(7) doxorubicin hydrochloride;

(8) enasidenib mesylate;

(9) gemtuzumab ozogamicin; (10) gilteritinib fumarate;

(11) idarubicin hydrochloride;

(12) ivosidenib;

(13) midostaurin;

(14) mitoxantrone hydrochloride;

(15) thioguanine;

(16) venetoclax;

(17) vincristine sulfate;

(18) a combination of cytarabine, daunorubicin hydrochloride, and etoposide phosphate (ADE);

(19) an mRNA demethylase inhibitor;

(20) an activator of METTL3 or METTL14 selected from the group consisting of methylpiperidine-3-carboxylate hydrochloride and methylpiperazine-2- carboxylate;

(21) brequinar sodium;

(22) a hypomethylating agent selected from the group consisting of azacytidine and decitabine; and

(23) all-frans-retinoic acid.

[0021] In methods according to the present invention, the bisantrene can inhibit expression of immune checkpoint gene LILRB4. In methods according to the present invention, the bisantrene can sensitize AML cells to T-cell cytotoxicity.

[0022] The method can further comprise the step of determining MRD in the patient. The determination of MRD can be determined prior to initiation of treatment or, alternatively, during treatment as a marker of the progress of treatment. The MRD status can be determined by next-generation sequencing, by multiparameter flow cytometry, or by a combination of both next-generation sequencing and multiparameter flow cytometry. [0023] In another alternative, the method can further comprise detecting of FLT3 mutations in the patient, or detecting other mutations such as DNMT3A mutations or a mutation selected from the group consisting of a mutation in TET2, a mutation in ASXL1, and a mutation in NPM1 in the patient.

[0024] Yet another aspect of the invention is a method for treatment of AML comprising:

(1 ) high intensity induction therapy comprising administering a therapeutically effective quantity of an anti-neoplastic agent for treatment of AML other than bisantrene to a patient to produce complete remission;

(2) consolidation of the AML resulting from the high intensity induction therapy of step (1);

(3) determining the presence or absence of MRD subsequent to step

(2);

(4) if MRD is found to be present in step (3), administering a therapeutically effective quantity of bisantrene to convert the patient to MRD-negative status;

(5) if MRD is found to be absent in step (3), performing a hematopoietic stem cell transplant leading to cure; and

(6) subsequent to the administration of the therapeutically effective quantity of bisantrene to convert the patient to MRD-negative status, performing of a hematopoietic stem cell transplant leading to cure.

[0025] Still another aspect of the invention is a method for treatment of AML comprising:

(1 ) performing high intensity induction therapy comprising administering a therapeutically effective quantity of an anti-neoplastic agent for treatment of AML other than bisantrene to a patient to produce complete remission;

(2) determining the presence or absence of MRD in the patient following step (1); (3) if MRD is found to be present in step (2), administering a therapeutically effective quantity of bisantrene;

(4) determining the presence or absence of MRD in the patient following step (3);

(5) consolidation of the AML subsequent to step (2) if MRD is found to be absent in step (2) or subsequent to step (4) if MRD is found to be present in step (4); and

(6) performing a hematopoietic stem cell transplant leading to cure subsequent to step (5) or subsequent to step (4) if MRD is found to be absent in step (4) leading to cure.

[0026] Yet another aspect of the invention is a pharmaceutical composition formulated for the treatment of MRD comprising:

(1 ) a therapeutically effective quantity of bisantrene; and

(2) at least one pharmaceutically acceptable excipient.

[0027] The pharmaceutically acceptable excipient can be, but is not limited to, a pharmaceutically acceptable excipient selected from the group consisting of:

(i) a liquid carrier;

(ii) an isotonic agent;

(iii) a wetting or emulsifying agent;

(iv) a preservative;

(v) a buffer;

(vi) an acidifying agent;

(vii) an antioxidant;

(viii) an alkalinizing agent;

(ix) a carrying agent;

(x) a chelating agent;

(xi) a coloring agent;

(xii) a complexing agent;

(xiii) a solvent; (xiv) a suspending and/or viscosity-increasing agent;

(xv) an oil;

(xvi) a penetration enhancer;

(xvii) a polymer;

(xviii) a stiffening agent;

(xix) a protein;

(xx) a carbohydrate;

(xxi) a bulking agent; and (xxii) a lubricating agent.

[0028] The pharmaceutical composition can comprise a therapeutically effective quantity of at least one additional agent for the treatment of AML as described above.

[0029] The additional agent for the treatment of AML can be, but is not limited to:

(1) arsenic trioxide;

(2) daunorubicin hydrochloride;

(3) cyclophosphamide;

(4) cytarabine;

(5) glasdegib maleate;

(6) dexamethasone;

(7) doxorubicin hydrochloride;

(8) enasidenib mesylate;

(9) gemtuzumab ozogamicin;

(10) gilteritinib fumarate;

(11) idarubicin hydrochloride;

(12) ivosidenib;

(13) midostaurin;

(14) mitoxantrone hydrochloride;

(15) thioguanine;

(16) venetoclax; (17) vincristine sulfate;

(19) an mRNA demethylase inhibitor;

(21) brequinar sodium;

(23) all-frans-retinoic acid.

[0030] The composition can further comprise a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L- lactide) micelles and b-cyclodextrin.

[0031] In another alternative, the pharmaceutical composition comprises a liposome.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following invention will become better understood with reference to the specification, appended claims, and accompanying drawings, where:

[0033] Figure 1 is a graph showing two-year survival and residual disease status at transplant for AML patients.

[0034] Figure 2 shows Measurable Residual Disease in peripheral blood after the second cycle of chemotherapy and clinical outcomes. Shown are the rates of overall survival (Panel A) and the cumulative incidence of relapse in all patients (Panel B), in those without FLT3- ITD mutations (Panel C) and those with FLT3-YTD mutations (Panel D), and in those without DNMT3A mutations (Panel E) and those with DNMT3A mutations (Panel F) among patients who were found to have measurable residual disease (MRD-positive) or no measurable residual disease (MRD-negative) in peripheral-blood samples.

[0035] Figure 3 shows the association between pretransplant disease status and outcome for patients with acute myeloid leukemia (AML) after myeloablative hematopoietic cell transplantation (HCT). Estimates of (A) overall survival, (B) progression-free survival, (C) cumulative incidence of relapse, and (D) cumulative incidence of nonrelapse mortality (NRM) after myeloablative allogeneic HCT for adults with AML, shown individually for patients in measurable residual disease (MRD) - negative (n = 235) and MRD- positive (n = 76) morphologic remission as well as those with active AML (n = 48).

[0036] Figure 4 shows relapse among patients with non-DTA mutations.

[0037] Figure 5 shows the rate of relapse according to results of next-generation sequencing and multiparameter flow cytometry.

[0038] Figure 6 shows bisantrene monotherapy trial design to improve MRD(-) status before HCT. (A) post-consolidation model; (B) second induction model.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Administration of bisantrene is a potential solution to the problem of MRD and the resulting poor survival rate of MRD-positive AML patients even following induction chemotherapy and HSCT. [0040] Bisantrene is an unusual agent with direct cytotoxic action as well as genomic and immunologic methods of action. The chemical name for bisantrene is 9, 10-anthracenedicarboxaldehyde-bis [(4, 5-dihydro-1 H-imidazole-2-yl) hydrazine] dihydrochloride, and it was originally classed as an anthracycline chemotherapeutic agent. As used herein, the term “bisantrene” refers to any pharmacologically compatible salt form, not only bisantrene dihydrochloride, unless the dihydrochloride or another specific pharmacologically compatible salt form is specifically indicated; however, bisantrene dihydrochloride is generally the preferred salt form in most pharmacological applications. These are drugs with planar structures based around a resonant aromatic ring structure that intercalates within the helices of DNA and disrupt various functions, including replication, presumably due to a strong inhibitory effect on the enzyme topoisomerase II. It was found that, like other anthracyclines, it could kill tumor cells in clonogenic assays and intercalate with DNA, where it inhibits both DNA and RNA synthesis. The primary chemotherapeutic mechanism for bisantrene is its preferential binding to A-T rich regions where it effects changes to supercoiling and initiates strand breaks in association with DNA associated proteins. This results from the inhibition of the enzyme topoisomerase II, which relaxes DNA coiling during replication. It was found that while inactive orally, intravenously (i.v.), intraperitoneally (i.p.), or subcutaneously (s.c.), the drug was effective in cancer models using colon 26, Lewis lung, Ridgway osteosarcoma, B16, Lieberman plasma cell, P388 or L1210 cancer cells. Activity in clonogenic assays from 684 patients was seen in breast, small cell lung, large cell lung, squamous cell lung, ovarian, pancreatic, renal, adrenal, head and neck, sarcoma, gastric, lymphoma and melanoma tumor cells, but not in colorectal cancer. Importantly, a lack of cross resistance with Adriamycin and mitoxantrone was found.

[0041] Bisantrene may have immunologic and/or genomic properties that might be responsible for some of its activities, and which may make this agent a useful tool in the combinatorial treatment of cancer in conjunction with newer immunotherapeutic agents. Subsequent to treatment with bisantrene, treated with bisantrene, and for 4 weeks thereafter, macrophages could be isolated from peritoneal exudate that had cytostatic anti-proliferative functionality in cultures of P815 (mastocytoma) tumor cells. Moreover, the supernatants from bisantrene activated macrophages also had a protective cytostatic effect in the tumor cell cultures. Further work revealed that macrophages activated with bisantrene and adoptively transferred to mice with EL-4 lymphomas more than doubled their median survival time, with 7 of 10 mice in the group being cured. Multiple administrations of activated macrophages were more effective than a single administration.

[0042] There is also evidence that the survivin inhibitors research that looked at the effect of bisantrene on survivin reported an interaction; one paper did find that the survivin inhibitors NSC80467 and YM155 acted in a manner that correlated with the known mechanism of DNA expression inhibition of bisantrene.

[0043] Bisantrene has also been found to have non-immunologic telomeric effects. Bisantrene binds to DNA at a site called a G-quadruplex, in which 4 guanines are associated by folding. Stabilization of the G-quadruplex can interfere with telomere- telomerase interaction and thus inhibit the activity of telomerase in various ways, including the displacement of telomerase binding proteins. Since the level of topoisomerase II inhibition does not always correlate with cytotoxic efficacy, alternative mechanisms may play a role in the actions of bisantrene. Analogs of bisantrene have been made in an attempt to improve upon the anti-telomerase activity; these analogs are described further below. Human melanoma (SK-Mel5) and colon cancer (LoVo) tumor cells were observed to lose their proliferative ability in the presence of these agents. Apoptosis was not observed; however a loss of immortality was seen, with treated cells reacquiring the ability to become senescent, age, and die.

[0044] As detailed above, in addition to direct antineoplastic effects related to the activity of bisantrene as a DNA intercalator, bisantrene also possesses other mechanisms of action, including immunopotentiation. These mechanisms are described in: (i) N.R. West et al. , “Tumor-Infiltrating Lymphocytes Predict Response to Anthracycline-Based Chemotherapy in Estrogen-Resistant Breast Cancer,” Breast Cane. Res. 13: R126 (2011), which concludes that the level of tumor-infiltrating lymphocytes is correlated with a response to the administration of anthracycline-based agents; the markers associated with tumor-infiltrating lymphocytes (TIL) include CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL; (ii) L. Zitvogel et al., “Immunological Aspects of Cancer Chemotherapy,” Nature Rev. Immunol. 8: 59-73 (2008), which states that DNA damage, such as that produced by intercalating agents such as bisantrene, induces the expression of NKG2D ligands on tumor cells in an ATM-dependent and CHK1 -dependent (but p53-independent) manner; NKG2D is an activating receptor that is involved in tumor immunosurveillance by NK cells, NKT cells, gd T cells and resting (in mice) and/or activated (in humans) CD8⁺ T cells, and also states that anthracycline-based agents may act as immunostimulators, particularly in combination with IL-12; such agents also promote HMGB1 release and activate T cells;

(iii) D.V. Krysko et al., “TLR2 and TLR9 Are Sensors of Apoptosis in a Mouse Model of Doxorubicin-Induced Acute Inflammation,” Cell Death Different. 18: 1316-1325 (2011), which states that anthracycline-based antibiotics induce an immunogenic form of apoptosis that has immunostimulatory properties mediated by MyD88, TLR2, and TLR9;

(iv) C. Ferraro et al., “Anthracyclines Trigger Apoptosis of Both G0-G1 and Cycling Peripheral Blood Lymphocytes and Induce Massive Deletion of Mature T and B Cells,” Cancer Res. 60: 1901-1907 (2000), which stated that anthracyclines induce apoptosis and ceram ide production, as well as activate caspase-3 in resting and cycling cells; the apoptosis induced is independent from CD95-L/CD95 and TNF/TNF-R; and (v) K. Lee et al., “Anthracycline Chemotherapy Inhibits HIF-1 Transcriptional Activity and Tumor- Induced Mobilization of Circulating Angiogenic Cells,” Proc. Natl. Acad. Sci. USA 106: 2353-2358 (2009), which provides another antineoplastic mechanism for anthracycline- based antibiotics, namely inhibition of HIF-1 mediated gene transcription, which, in turn, inhibits transcription of VEGF required for angiogenesis; HIF-1 also activates transcription of genes encoding glucose transporter GLUT1 and hexokinases HK1 and HK2, which are required for the high level of glucose uptake and phosphorylation that is observed in metastatic cancer cells, and pyruvate dehydrogenase kinase 1 (PDK1), which shunts pyruvate away from the mitochondria, thereby increasing lactate production; patients with HIF-1 a overexpression based on immunohistochemical results were suggested to be good candidates for treatment with anthracycline-based antibiotics.

[0045] Several clinical trials have investigated the pharmacokinetics of bisantrene in humans. In one trial of patients given a 90 min infusion at 260 mg/m² a biphasic elimination with an initial half-life of 65±15 min, a terminal half-life of 1142±226 min, and a steady state volume of distribution (Vdss) of 1845 L/m² Plasma clearance in this trial was 735 ml_/min/m², with 11.3% of the administered dose excreted unchanged in the urine in 24 hr. In another trial, doses of 80-250 mg/m² were assessed, and the initial and terminal half-lives were 0.6 hr and 24.7 hr, respectively, with a clearance of 1045.5±51.0 mL/kg/hr and a calculated volume of distribution of 42.1 ±5.9 L/kg. In this study only 3.4±1.1% of the administered dose was found in the urine over 96 hr. In three other single dose studies triphasic elimination was reported, one with t½a, b, and g of 3.44 min, 1.33 hr and 26.13 hr, respectively, another was 3 min, 1 hr, and 8 hr respectively, and the last revealed clearances of 0.1 hr, 1.9 hr and 43.9 hr, respectively. In one report a large volume of distribution (687 L/m²) was interpreted as tissue sequestration of the drug with a subsequent depot effect. In a 72- hr infusion study, a plasma concentration of 12±6 ng/mL was observed at a dose of 56 mg/m², while a dose of 260 mg/m² resulted in a plasma concentration of 55±8 ng/mL.

In this trial plasma clearance was 1306±179 mL/min/m² with urinary excretion of 4.6% of the dose in 24 hr. Finally, in another study, a 5 day schedule of 60 min infusions revealed a t ½ a and b of 0.9 and 9.7 hr, respectively with 7.1 % of the dose excreted in the urine.

[0046] The structure of bisantrene dihydrochloride is shown in Formula (I)

(I).

[0047] Bisantrene is a tricyclic aromatic compound with the chemical name, 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1 H-imidazol-2-yl)hydrazine] dihydrochloride. The molecular formula is C22H22N8 · 2HCI and the molecular weight, 471.4. The alkylimidazole side chains are very basic and, at physiologic pH, are positively charged. This is believed to facilitate electrostatic attractions to negatively charged ribose phosphate groups in DNA.

[0048] Bisantrene is typically administered intravenously, either centrally or peripherally.

[0049] Various formulations suitable for use in the administration of bisantrene or derivatives or analogs thereof are known in the art. United States Patent No. 4,784,845 to Desai et al. discloses a composition of matter for delivery of a hydrophobic drug (i.e. , bisantrene or a derivative or analog thereof) comprising: (i) the hydrophobic drug; (ii) an oleaginous vehicle or oil phase that is substantially free of butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT); (iii) a co-surfactant or emulsifier; (iv) a co-surfactant or auxiliary emulsifier; and (v) benzyl alcohol as a co solvent. United States Patent No. 4,816,247 by Desai et al. discloses a composition of matter for delivery by intravenous, intramuscular, or intraarticular routes of hydrophobic drugs (such as bisantrene or a derivative or analog thereof) comprising: (i) the hydrophobic drug; (ii) a pharmaceutically acceptable oleaginous vehicle or oil selected from the group consisting of: (a) naturally occurring vegetable oils and (b) semisynthetic mono-, di-, and triglycerides, wherein the oleaginous vehicle or oil is free of BHT or BHA; (iii) a surfactant or emulsifier; (iv) a co-surfactant or emulsifier; (v) an ion-pair former selected from C6-C20 saturated or unsaturated aliphatic acids when the hydrophobic drug is basic and a pharmaceutically acceptable aromatic amine when the hydrophobic drug is acidic; and (vi) water. United States Patent No. 5,000,886 to Lawter et al. and United States Patent No. 5,143,661 to Lawter et al. disclose compositions for delivery of pharmaceutical agents such as bisantrene or a derivative or analog thereof comprising a microcapsule, wherein the microcapsule includes a hardening agent that is a volatile silicone fluid. United States Patent No. 5,070,082 to Murdock et al. , United States Patent No. 5,077,282 to Murdock et al., and United States Patent No. 5,077,283 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs, including bisantrene and derivatives and analogs, that are salts of a phosphoramidic acid. United States Patent No. 5,116,827 to Murdock et al. and United States Patent No. 5,212,291 to Murdock et al. disclose prodrug forms of poorly soluble hydrophobic drugs, including bisantrene and derivatives and analogs, that are quinolinecarboxylic acid derivatives. United States Patent No. 5,378,456 to Tsou includes compositions containing an anthracene antitumor agent, such as bisantrene or a derivative or analog thereof, in which the bisantrene or derivative or analog thereof is conjugated to or admixed with a divinyl ether-maleic acid (MVE) copolymer. United States Patent No. 5,609,867 to Tsou discloses polymeric 1 ,4-bis derivatives of bisantrene and copolymers of bisantrene and another monomer, such as a dianhydride.

[0050] Alternatively, methods and compositions described herein can use a derivative or analog of bisantrene in place of bisantrene itself. Derivatives and analogs of bisantrene are described in PCT Patent Application Publication No. WO 2015/013581 by Garner et al. Additional derivatives and analogs of bisantrene are disclosed in R. Su et al., “Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion,” Cancer Cell 38: 1-18 (2020) (“Su et al. (2020)”). These derivatives and analogs are shown below as Formulas (A-l), (A-ll), (A-lll), (A-IV), (A-V), and (A-VI):

(A-l); (A- IV);

(A-VII). [0051] Accordingly, one aspect of the invention is a method of converting AML patients, subsequent to initial treatment, from MRD(+) status to MRD(-) status by administration of a therapeutically effective quantity of bisantrene, or, in an alternative, a therapeutically effective quantity of a derivative or analog of bisantrene having appropriate anti-neoplastic activity to the patient. Typically, bisantrene is administered. Preferably, the bisantrene is administered as bisantrene dihydrochloride.

[0052] Patients with AML are currently split into “older” or “younger” patients by oncologists on the basis of age and other co-morbidities that predict if the patient can survive the rigors of induction chemotherapy. “Older” patients receive palliative care and/or low dose hypomethylating agents (azacytidine or decitabine) or low dose cytarabine (LDAC). The aim is not to cure the disease, but merely to slow the disease progression in the patient.

[0053] In “younger” patients the aim is to achieve a complete remission (CR) via the use of high-intensity induction (HIT) chemotherapy, followed by consolidation before hematopoietic cell transplantation (HSCT). In the absence of achieving a CR the patient may undergo multiple rounds of induction or consolidation therapy with the aim of achieving a CR before HCT or may undergo HSCT even in the absence of a CR.

[0054] CR in AML is defined by the patient having less than 5% blasts observed in bone marrow biopsy after induction and/or consolidation. While CR is predictive of a cure after HCT, many patients still relapse despite being in CR at the time of transplant. While the genotype of the AML plays an important role (some AML subtypes are more aggressive and resistant to current treatments), the role of residual disease in CR patients has been found to be of critical importance for predicting relapse-free survival (E.H. Estey, “Acute Myeloid Leukemia: 2019 Update on Risk-Stratification and Management,” Am. J. Hematol. 93: 1267-1291 (2018); G.J. Schuurhuis et al. , “Minimal/Measurable Residual Disease in AML: a Consensus Document from the European LeukemiaNet MRD Working Party,” Blood 131: 1275-1291 (2018).

[0055] In methods according to the present invention for treatment of AML subsequent to induction chemotherapy, as described above, suitable dosages of bisantrene (or, alternatively, a derivative or analog of bisantrene) can be determined by one of ordinary skill in the art. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular therapeutic agent, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the severity of the condition, other health considerations affecting the subject, and the status of liver and kidney function of the subject. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic agent employed, as well as the age, weight, condition, general health and prior medical history of the subject being treated, and like factors. Methods for determining optimal dosages are described in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed.,

2000. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage-determination tests in view of the experimental data for an agent.

[0056] However, typically, administration of bisantrene in methods according to the present invention is performed for 7 days at a dosage from about 200 mg/m²/day to about 300 mg/m²/day. Preferably, administration of bisantrene in methods according to the present invention is performed for 7 days at a dosage from about 225 mg/m²/day to about 275 mg/m²/day. More preferably, administration of bisantrene in methods according to the present invention is performed for 7 days at a dosage of about 250 mg/m²/day. In other alternatives, the period over which the bisantrene can be administered can be varied, for example, such as for 5 days, 6 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. [0057] The bisantrene can be administered as a drug compound or as a component of a pharmaceutical composition. Suitable pharmaceutical compositions are described in PCT Patent Application Publication No. WO 2015/013581 by Garner et al.

In general, such pharmaceutical compositions include at least one pharmaceutically acceptable excipient as described below. In some alternatives, the bisantrene can be administered in a liposome, as described in PCT Patent Application Publication No. WO 2019/073296 by Rothman.

[0058] As another alternative, the bisantrene (or in some cases, the derivative or analog of bisantrene) can be administered together with a therapeutically effective quantity of at least one additional therapeutic agent for treating AML. Therapeutic agents that have been approved for treating AML include: arsenic trioxide; daunorubicin hydrochloride; cyclophosphamide; cytarabine; glasdegib maleate; dexamethasone; doxorubicin hydrochloride; enasidenib mesylate; gemtuzumab ozogamicin; gilteritinib fumarate; idarubicin hydrochloride; ivosidenib; midostaurin; mitoxantrone hydrochloride; thioguanine; venetoclax; vincristine sulfate; and a combination of cytarabine, daunorubicin hydrochloride, and etoposide phosphate (ADE). Other alternatives for additional therapeutic agents are described below.

[0059] Arsenic trioxide is a cytostatic agent particularly useful in the treatment of the refractory promyelocytic (M3) subtype of AML. Daunorubicin hydrochloride is a DNA intercalator and topoisomerase II inhibitor. Cyclophosphamide is an alkylating agent that crosslinks DNA. Cytarabine is a nucleoside analog that acts as an antimetabolite. Glasdegib maleate is a small molecule inhibitor of the sonic hedgehog receptor smoothened (SMO). Dexamethasone is a corticosteroid that has a direct antineoplastic effect against AML as well as controlling certain inflammation-related side effects of other antineoplastic agents. Doxorubicin hydrochloride is a DNA intercalator and topoisomerase II inhibitor. Enasidenib mesylate acts by decreasing total levels of the ( R ) stereoisomer of 2-hydroxyglutarate. Gemtuzumab ozogamicin is a monoclonal antibody to CD33 linked to a cytotoxic agent from the class of calicheamicins.

Gilteritinib fumarate is an inhibitor of AXL receptor tyrosine kinase. Idarubicin hydrochloride is a DNA intercalator and topoisomerase II inhibitor. Ivosidenib is an inhibitor of the IDH1 isocitrate dehydrogenase enzyme. Midostaurin is a multi-targeted protein kinase inhibitor. Mitoxantrone hydrochloride is an anthracenedione derivative that is a topoisomerase II inhibitor. Thioguanine is a base analog that inhibits the synthesis of guanine-containing nucleotides. Venetoclax is a BH3 mimetic that induces apoptosis. Vincristine sulfate is a small molecule that binds to tubulin, inhibiting mitosis. Etoposide phosphate, a component of ADE, is a topoisomerase II inhibitor that causes breakage of DNA strands and subsequent apoptosis.

[0060] Suitable dosages, dosage frequencies, dosage durations, and routes of administration for these additional agents, as well for additional agents described below, are known in the art. These additional agents can either be administered simultaneously with the bisantrene or the derivative or analog of bisantrene, or at a different time than the bisantrene or the derivative or analog of bisantrene. If the additional agent is administered at a different time than the bisantrene or the derivative or analog of bisantrene, it can either be administered before or after the bisantrene or the derivative or analog of bisantrene. One of ordinary skill in the art can determine a suitable schedule for administration based on variables such as the age, weight, and sex of the patient, the severity of the AML, including the presence or absence of MRD, genetic markers such as further described below, and pharmacokinetic parameters such as liver and kidney function.

[0061] In still another alternative, the bisantrene can be administered together with a therapeutically effective quantity of an inhibitor of oncogenic FTO demethylase (Y. Huang et al. , “Small Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia,” Cancer Cell 35: 677-691 (2019) (“Huang et al. (2019)”). RNA epitranscriptomics represents a recently identified layer of regulation of genetic information. A^-methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic mRNA and also in noncoding RNAs. The protein fat-mass- and obesity associated protein (FTO) has been identified as an m⁶A demethylase corroborates that the m⁶A modification is a dynamic process; m⁶A is relatively enriched near stop codons, 3'-UTRs (untranslated regions), as well as coding regions; the presence of m⁶A is considered to be critical for the regulation of mRNA stability, splicing, transport, translation, primary microRNA processing, and protein-RNA interactions. The m⁶A levels depend on the functional interplay among several proteins. METTL3 and METTL14, the m⁶A methyltransferases, form a heterodimer with the support of cofactors to induce m⁶A methylation. The two demethylases FTO and ALKBH5, which belong to the Fe²⁺- and 2-oxoglutarate (20G)-dependent AlkB dioxygenase family, primarily catalyze m⁶A demethylation, although FTO was initially identified as a demethylase of A/³-methylthymidine and A/³-methyluridine in vitro and can also catalyze the demethylation of A/^'-O-dimethyladenosine (m⁶A_m).

[0062] The discovery of small-molecule inhibitors of FTO enabled temporal intervention of mRNA methylation. Rhein has been identified as an FTO inhibitor.

Rhein is an anthraquinone derivative with the structure 4, 5-di hydroxy-9, 10- dioxoanthracene-2-carboxylic acid. Another FTO inhibitor is the non-steroidal anti inflammatory agent meclofenamic acid, shown below as Formula (F-l), and its ethyl ester, shown below as Formula (F-ll):

(F-ll). Fluorescein also both inhibits and labels FTO. Additional FTO inhibitors are disclosed in J.D.W. Toh et al. , “A Strategy Based on Nucleotide Specificity Leads to a Subfamily- Selective and Cell-Active Inhibitor of A^-Methyladenosine Demethylase FTO,” Chem. Sci. 6: 112-122 (2015), including the compounds shown below as Formulas (F-lll) and (F-IV):

(F-IV);

(F-IV) is an ethyl ester of (F-lll). Additionally, the R-enantiomer of 2-oxoglutarate was demonstrated to possess anti-proliferative activity against acute myeloid leukemia cells, primarily by targeting FTO.

[0063] The dysfunction of FTO demethylation has been associated with human diseases, especially malignancy. The ethyl ester of meclofenamic acid (Formula F-ll) has been shown to suppress glioblastoma stem cell-initiated tumorigenesis. Rhein or meclofenamic acid could be uniquely positioned to either prevent or override tyrosine kinase inhibitor resistance by inhibiting FTO demethylation with respect to a subset of mRNA.

[0064] Huang et al. (2019) described the synthesis and activity of a derivative of meclofenamic acid entitled FB23. This compound is shown below as Formula (F-VI): (F-VI).

[0065] The FB23 derivative of meclofenamic acid bound tightly to the substrate binding site of FTO, and the complementarity between the FB23 derivative and FTO precludes nonspecific binding to either RNA demethylase ALKBFI5 or the DNA repair enzymes ALKBFI2 or ALKBFI3. In addition, extra hydrogen bonding was observed between nitrogen or oxygen in the extended heterocyclic ring of FB23 and the amide backbone of Glu234 of FTO, which likely further enhances the inhibitory activity of FB23.

[0066] According to Fluang et al. (2019), FB23 only exhibited moderate antiproliferative effects, likely to its poor cellular uptake. Therefore, Huang et al. (2019) prepared derivatives of the benzylcarboxylic acid based on the principles of bioisosterism. One of these compounds, FB23-2, displayed increased antiproliferative effects. This further derivative compound is shown below as Formula (F-VII):

(F-VII).

[0067] FB23-2 was shown to increase RNA methylation in a panel of AML cells; it is likely that m⁶A is the main substrate of FTO in AML cells. FB23-2 also displayed a high degree of selectivity toward FTO. For FB23-2, there was minimal inhibitory activity on other potential epigenetic targets involved in AML or other malignancies, including the histone demethylases, disruptor of telomere silencing 1-like proteins, bromodomain- containing reader proteins, lysine-specific demethylase-1 , and Jumonji domain- containing histone demethylases. This derivative also barely inhibited the oncogenic proteases. Neither FB23 nor FB23-2 was observed to significantly inhibit cyclooxygenases even at relatively high concentrations.

[0068] The compound FB23-2 was shown to exhibit FTO-dependent antiproliferative activity and also was shown to promote myeloid differentiation and apoptosis. In general, FB23-2 caused effects similar to inhibition of FTO activity by FTO knockdown such as shFTO. All of FTO knockdown, FB23, and FB23-2 had relatively similar activity; FTO knockdown and inhibition stimulated apoptosis and the p53 pathway, while repressing MYC targets, G2M checkpoints, and E2F targets. The activation of apoptosis and p53 pathways was considered to be through an m⁶A- dependent mechanism. Therefore, regulation of m⁶A was considered to act as the major effector of FB23-2 in AML cells.

[0069] According to Huang et al. (2019), the FB23-2/FTO axis-induced upregulation of RARA and ASB2 and downregulation of MYC and CEBPA might depend on an increased abundance of m⁶A in mRNA. This supports the conclusion that regulation of m⁶A acts as the major effector of FB23-2 in AML cells; retention of m⁶A in those cells supports apoptosis and inhibition of cell proliferation in these cells.

[0070] Furthermore, according to Huang et al. (2019), FB23-2 appears to be safe in mice and displays a favorable pharmacokinetic profile. The data indicated that FB23-2 in a dosage of 20 mg/kg was safe for determining in vivo efficacy. Regarding the pharmacokinetic profile of the compound, based on administration of a single dose of 3 mg/kg intraperitoneally to Sprague-Dawley rats. The Cmax and Tmax value of FB23-2 were 2,421.3+90.9 ng/mL and 0.08 h, respectively. FB23-2 elimination half-life, T1/2 was 6.7+1.3 h, and the AUCo-24 was 2,184+152 hxng/mL. Meanwhile, FB23 was also detected, with Cmax and Tmax as 142.5+26.1 and 0.4+0.1 h, respectively. The metabolic stability of FB23-2 in the SD rat liver microsome was also determined, with an estimated T1/2 of 128 min, and an intrinsic clearance of 19.7 mL/min/kg. The authors also measured the degree of protein binding by FB23-2. Nearly 100% FB23-2 inhibitor was bound to plasma proteins. In summary, FB23-2 displayed a favorable pharmacokinetic profile for in vivo study.

[0071] Fluang et al. (2019) also assessed the therapeutic effects of FB23-2 in vivo with a xenotransplantation leukemic model. NOD/LtSz-scid IL2RG-SGM3 (NSGS) mice were xenotransplanted with MONOMAC6 AML cells, and 10 days post xenotransplantation, FB23-2 (2 mg/kg) or vehicle control was intraperitoneally injected into the individual mice daily for 10 days. Notably, FB23-2 injection substantially delayed the onset of full-blown leukemic symptoms and significantly prolonged survival by almost doubling the median survival. Compared with the vehicle, FTO inhibitor treatment suppressed leukemia malignancy, including reduced splenomegaly and hepatomegaly. Fluorescence-activated cell sorting (FACS) analysis confirmed that FB23-2 injection suppressed the abundance of human AML cells in the recipient mice. To further interpret the effect of FB23-2 on differentiation of AML cells in vivo, the authors collected peripheral blood (PB), BM, and spleen samples of FB23-2- and vehicle control-treated xenograft mice and stained them with anti-human CD15 and anti human CD11b antibodies. As determined by FACS, FB23-2 treatment promoted AML cell differentiation in vivo. Wright-Giemsa staining of PB smears revealed that leukemic blasts from FTO inhibitor-treated AML mice were inhibited and partially differentiated; consistently, haematoxylin-eosin (H&E) staining of spleen and liver also showed less AML cell dissemination in FB23-2 -treated mice. Taken together, the data provided in Huang et al. (2019) suggest that pharmacological inhibition of FTO by FB23-2 substantially suppresses leukemia progression and prolongs survival. [0072] Furthermore, according to Huang et al. (2019), the therapeutic potential of FB23-2 was assessed in treating human primary AML cells. Four AML patients with diverse cytogenetics were tested. FB23-2 suppressed proliferation of all four sets of primary AML cells, with IC50 values ranging from 1.6 to 16 mM. FB23-2 also induced cell apoptosis, decreased colony-forming unit (CFU) capacity, and accelerated all -trans- retinoic acid (ATRA)-mediated myeloid differentiation of these primary AML cells. Furthermore, FB23-2 treatment also upregulated the expression of both ASB2 and RARA, two direct targets of FTO, and elevated global mRNA m⁶A abundance, thus supporting the conclusion that FB23-2 displays therapeutic effects via directly targeting FTO signaling in patient-derived primary AML cells.

[0073] Additionally, according to patient-derived xenotransplantation (PDX) AML mouse model. Primary AML cells were xenotransplanted into sublethally irradiated NSGS mice. The authors monitored the engraftment of AML leukemia cells in vivo by FACS analysis of the percentage of donor AML cells in PB in recipient mice. When recipient mice had 3%-5% donor-derived AML cells, the recipient mice were treated with FB23-2 or DMSO (a vehicle as a control) for 17 days. The disease latency of FB23-2- treated mice (median survival time of 58 days) was significantly prolonged compared with that of control mice (median survival time of 48 days). Furthermore, FACS analysis of engrafted AML cells in recipient mice revealed a significantly reduced proportion of AML blast cells in peripheral blood (PB) and bone marrow (BM) upon FB23-2 treatment. Consistent with the findings that FB23-2 induced differentiation of AML cell lines in vitro, the authors concluded that more differentiated myeloid cells with an increased ratio of cytoplasm/nucleus were present in FB23-2-treated mice. The leukemia cells from FB23-2 -treated PDX mice gave rise to significantly fewer CFUs with markedly reduced sizes of colonies than the leukemia cells from the DMSO-treated PDX mice did, thus suggesting that the leukemia malignancy of FB23-2-treated AML cells was significantly impaired. Notably, not only were the bulk AML cells affected, but also leukemia stem cells (LSCs, defined by CD34⁺CD38 ) were significantly eliminated by FB23-2 in vivo in the treated mice. [0074] Additionally, according to Huang et al. (2019), to further evaluate the number of functional LSCs in primary PDX mice, the authors performed a secondary transplantation. The secondary recipients of AML cells from primary DMSO-treated PDX mice (control) had markedly higher engraftment compared with the secondary recipient mice with AML cells from primary FB23-2 -treated PDX mice. All of the control, secondary PDX mice died within 66 days while 50% of the secondary PDX mice with FB23-2 -treated AML cells still survived after 100 days, thus suggesting that the number of functional LSCs that are able to regenerate leukemia in vivo in secondary recipients was significantly reduced after FB23-2 treatment in the primary recipient mice. Taken together, that data indicate that FB23-2-induced differentiation of AML cells significantly reduced the number of functional primary AML LSCs in vivo.

[0075] In conclusion, Huang et al. (2019) stated that epitranscriptomics is a rapidly evolving field, and that emerging evidence suggests that deregulation of m⁶A modification on RNA contributes to leukemogenesis. METTL3 and METTL14, the m⁶A methyltransferases, have been reported to control and/or maintain myeloid leukemia. Additionally, the m⁶A demethylase FTO has been found to play an oncogenic role in a subset of AMLs. Moreover, the compound R-2HG, by suppression of FTO activity, has been shown to exhibit significant antitumor effects in AML. Few inhibitors for regulation of RNA methylation have been characterized, which is in sharp contrast to the characterization of factors of DNAand histone epigenetics. The authors reported that through structure-based rational designs, they have successfully developed more effective small-molecule inhibitors of FTO. The meclofenamic acid (MA)-derived inhibitor FB23 displays significantly improved inhibitory activity on FTO demethylation of m⁶A-RNA/V? vitro. Next, the authors optimized the physicochemical properties of FB23, thus leading to the identification of FB23-2 with a significantly improved ability to hinder the proliferation of a panel of AML cell lines, and also inhibits primary AML LSCs in PDX mice, thus suggesting that FTO might serve as a potential molecular target in LSCs in order to inhibit leukemogenesis. The discovery of FB23-2 and its anti-proliferative effects on AML would increase the current intense interest in RNA methylation, especially with regard to its pharmacological applications. [0076] Accordingly, another aspect of the present invention comprises administration of a therapeutically effective quantity of an inhibitor of oncogenic FTO demethylase together with bisantrene or a derivative or analog of bisantrene as described above to treat measurable residual disease in acute myeloid leukemia. The inhibitor of oncogenic FTO demethylase can be selected from the group consisting of: rhein; meclofenamic acid; meclofenamic acid ethyl ester; a compound of Formula (F-lll) as described above; a compound of Formula (F-IV) as described above, an ethyl ester of the compound of Formula (F-lll); the R-enantiomer of 2-oxoglutarate; FB23, a derivative of meclofenamic acid; and FB23-2, a derivative of FB-23. Typically, the inhibitor of oncogenic FTO demethylase is FB23-2.

[0077] In still another alternative, the bisantrene can be administered together with a therapeutically effective quantity of an activator of METTL3 or METTL14 (S. Selberg et al., “Discovery of Small Molecules that Activate RNA Methylation Through Cooperative Binding to the METTL3-14-WTAP Complex Active Site,” Cell Rep. 26: 3762-3771 (2019)). These activators include methylpiperidine-3-carboxylate hydrochloride and methylpiperazine-2-carboxylate.

[0078] In yet another alternative, the treatment methods described above can be combined with a determination of MRD status. MRD status can be determined by both next-generation sequencing (NGS) and multiparameter flow cytometry (MFC); both methods can be used in combination (M. Jongen-Lavrencic et al., “Molecular Minimal Residual Disease in Acute Myeloid Leukemia,” N. Engl. J. Med. 378: 1189-1199 (2018); G.J. Schuurhuis et al. (2018), supra. Determination of MRD status can be made before the initiation of treatment or during treatment as a marker of progress of treatment.

[0079] Another alternative for treatment methods according to the present invention includes detection of FLT3 mutations, including internal tandem duplications (ITD) and mutations in the tyrosine kinase domain (TKD). FLT3- ITD mutations are considered driver mutations that present with a high leukemia burden and are associated with poor prognosis; the prognostic value of FLT3- TKD mutations is uncertain (N. Daver et al. , “Targeting FLT3 Mutations in AML: Review of Current Knowledge and Evidence,” Leukemia 33: 299-312 (2019)). Screening methods for detection of mutations in FLT3 include PCR plus denaturing HPLC (M. Bianchini et al., “Rapid Detection of Flt3 Mutations in Acute Myeloid Leukemia Patients by Denaturing HPLC.” Clin. Chem. 49: 1642-1650 (2003)).

[0080] Still another alternative for treatment methods according to the present invention includes detection of DNMT3A mutations (H.A. Hou et al., “DNMT3A Mutations in Acute Myeloid Leukemia: Stability During Disease Evolution and Clinical Implications,” Blood 112: 559-568 (2012)). These mutations cause dysregulated patterns of DNA methylation. Other mutations, including mutations in TET2, ASXL1, and NPM1 can also be detected and may be associated with variations in prognosis (D.P. Steensma & B.L. Ebert, “Clonal Hematopoiesis After Induction Chemotherapy for Acute Myeloid Leukemia,” N. Engl. J. Med. 378: 1244-1245 (2018); D.P. Steensma et al., “Clonal Hematopoiesis of Indeterminate Potential and Its Distinction from Myelodysplastic Syndrome,” Blood 126: 9-16 (2015)). Mutations other than mutations in DNMT3A, TET2, and ASXL1 are frequently referred to as “non-DTA mutations.”

[0081] These mutations can be detected by methods known in the art. Methods for DNA sequencing are known in the art. Methods for DNA sequencing include: the Maxam-Gilbert sequencing method involving chemical modification of DNA followed by cleavage at specific bases; the Sanger chain termination method; stepwise sequencing with removable 3'-blockers on DNA arrays; DNA colony sequencing involving random surface PCR-arraying methods; pyrosequencing; sequencing by synthesis; massively parallel signature sequencing; Polony sequencing; parallelized pyrosequencing; sequencing employing reversible dye-terminators; sequencing by use of rolling circle replication to amplify small fragments of DNA into DNA nanoballs; sequencing involving use of DNA fragments with added poly A tail adapters attached to a flow cell surface; nanopore DNA sequencing; sequencing employing tunneling currents; sequencing by hybridization; sequencing by mass spectroscopy; and sequencing employing RNA polymerase attached to polystyrene beads. Other DNA sequencing methods are known in the art. Other mutation analysis techniques are known in the art, including, but not limited to, restriction fragment length polymorphism (RFLP) analysis, terminal restriction fragment length polymorphism (TRFLP) analysis, cleaved amplified polymorphic sequence (CAPS) analysis, and other methods involving use of the polymerase chain reaction (PCR) procedure.

[0082] In still other alternatives, the bisantrene or the derivative or analog thereof can be administered in a formulation employing either mPEG-b-PLA micelles or b-cyclodextrin in order to improve bioavailability (R. Su et al. , “Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion,” Cancer Cell 38: 1- 18 (2020) (“Su et al. (2020)”).

[0083] According to Su et al. (2020), bisantrene (referred to as “CS1” in Su et al. (2020) and brequinar sodium (6-fluoro-2-(2'-fluoro[1 ,T-biphenyl]-4-yl)-3-methyl-4- quinolinecarboxylic acid sodium) (referred to as “CS2” in Su et al. (2020)) were found to be highly efficacious inhibitors of FTO with potent anti-leukemic efficacy in vitro compared with FB23-2 and MO-l-500 (A/-(3,4-dihydroxy-5-(4-chlorophenyl)-2- furanyl)ethanesulfonamide). These compounds were more effective in leukemia cell lines with high levels of FTO expression; knockdown of FTO expression reduced their sensitivity to these compounds. These results suggest that the anti-leukemia effects of CS1 and CS2 are FTO-abundance dependent. Both of these compounds significantly inhibited the viability of human primary AML cells, but largely spared healthy control cells. The direct interactions between CS1 and CS2 were confirmed by nuclear magnetic resonance. The specific interactions between these compounds and domains of FTO were further analyzed by drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA), which showed that residues H231 and E234 were essential for the binding of FTO with CS1. Both CS1 and CS2 efficiently suppressed the m⁶A demethylase activity of FTO, with IC50 values in the nanomolar range. Crosslinking immunoprecipitation-qPCR data confirmed that CS1 and CS2 block the binding of FTO with its target RNAs, including MYC, CEPBA, and RARA. Treatment with CS1 or CS2 also increased the global m⁶A abundance in AML cells, but had no effect on the quantity of FTO protein. However, neither CS1 nor CS2 treatment suppressed the enzymatic activity of ALKBH5, another major m⁶A demethylase, or TET1, another a-KG-dependent dioxygenase, highlighting the selectivity of CS1 and CS2 against FTO.

[0084] Additionally, pharmacological inhibition of FTO by CS1 or CS2 resulted in substantially increased apoptosis and cell-cycle arrest at the Go phase in human AML cells. Both inhibitors, either alone or together with all-frans-retinoic acid, also significantly promoted myeloid differentiation in human AML cells.

[0085] Leukemia stem/initiating cells (LSCs/LICs), characterized by their unlimited self-renewal potential, are considered to be the root cause of the treatment failure and relapse of AML; thus, eradication of LSCs/LICs is necessary to achieve a cure. Either knockdown of FTO or pharmacological inhibition of FTO resulted in a remarkable decrease of LSCs/LICs in murine AML models; 50 nM CS1 almost completely inhibited the repopulating capacity of AML cells.

[0086] RNA sequencing and qPCR data showed that CS1 or CS2 treatment substantially decreased MYC and CEBPA expression while increasing RARA and ASB2 expression, which are positive and negative targets of FTO, respectively. By targeting FTO, CS1 and CS2 also increased m⁶A abundance on FTO targeted RNAs, such as MYC and CEBPA mRNAs and small nuclear RNAs (snRNAs). Therefore, it was suggested that CS1 and CS2 exert their anti-leukemic effects through modulation of the essential signaling pathways of FTO.

[0087] In vivo, using a patient-derived xenotransplantation (PDX) AML model, although CS2 dramatically reduced leukemia infiltration and doubled the overall survival rate, CS1 was not as effective; this was attributed to poor solubility and uptake of CS1 in vivo. To improve the bioavailability of CS1 , the compound could be administered together with either mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles or b-cyclodextrin. The use of either of these alternatives provided potent anti- AML efficacy in a PDX AML model in vivo, accompanied by a significant impact on the expression of FTO targets. The administration of CS1 in these alternatives to increase bioavailability demonstrated the potent therapeutic efficacy of CS1 , administered with mPEG-b-PLA micelles or b-cyclodextrin, in treating AML, including relapsed AML. The dosages tested, which demonstrated therapeutic effects, were 5 mg/kg once every other day, 10 times.

[0088] Additionally, it was shown that the activity of wild-type FTO could increase the expression of immune checkpoint gene LILRB4. The increased expression of this gene is associated with the development of resistance to the therapeutic efficacy of hypomethylating agents (HMAs) such as azacytidine or decitabine in AML. Knockdown of FTO expression or administration of CS1 or CS2, however, significantly decreased the expression of LILRB4. The results show that FTO positively regulates LILRB4 expression in AML by suppression of the decay of m⁶A-modified LILRB4 mRNA induced by the activity of the m⁶A reader YTHDF2. The FTO/m⁶A axis regulates immune checkpoint gene expression, which, in turn, affects the development of resistance to hypomethylating agents.

[0089] In order to determine whether pharmacological inhibition of the FTO/m⁶A/LILRB4 axis could reprogram the immune response, the authors pretreated AML cells with CS1 or CS2 and then co-cultured them with activated T cells. FTO inhibition sensitized human AML cells to T cells, accompanied by decreased expression of LILRB4. FTO inhibition (by CS1 or CS2) synergized with T cell treatment and substantially suppressed AML progression, resulting in remarkably prolonged survival in the combinational treatment groups. Consistent with the role of FTO in mediating HMA- induced upregulation of immune checkpoint genes and subsequent immune evasion, FTO inhibition also synergized with HMAs such as azacytidine or decitabine in inhibiting AML progression in immune-competent BMT recipient mice, and the combinations showed much improved therapeutic efficacy than either treatment alone. Collectively, FTO inhibition could suppress immune checkpoint gene expression and thereby sensitize AML cells to T-cell cytotoxicity and also overcome HMA-induced immune evasion.

[0090] To evaluate the potential drug toxicity of CS1 and CS2 in vivo, the authors injected two doses for each compound (5 mg/kg/day (i.e. , the dose used for AML mouse treatment) and 20 mg/kg/day) into C57BL/6 mice once every other day for 20 days, and euthanized all the mice 10 days after the final treatment. No significant toxicities were observed.

[0091] FTO inhibitors may also have therapeutic utility in treatment of other malignancies, including glioblastoma, breast cancer, and pancreatic cancer.

[0092] Therefore, an additional aspect of the present invention is a method of converting an AML patient, subsequent to initial treatment, from MRD(+) status to MRD(-) status, comprising the step of administering a therapeutically effective quantity of bisantrene to the patient, wherein the bisantrene is administered in a formulation comprising a bioavailability enhancer selected from the group consisting of mPEG-b- PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b-cyclodextrin. In this aspect of the invention, the dosage of bisantrene can be about 5 mg/kg once every other day, 10 times; other alternatives for dosage, frequency of administration, and duration of administration are within the scope of the invention. The bisantrene can be administered in a formulation comprising a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b-cyclodextrin in other methods as described herein, including methods in which the bisantrene is administered subsequent to the performance of high intensity induction therapy, consolidation of the AML, and, if MRD is found to be present, administration of bisantrene. In such methods, the additional therapeutic agent for high intensity induction therapy can include one or more of brequinar sodium; a hypomethylating agent selected from the group consisting of azacytidine and decitabine; and all-frans-retinoic acid, in addition to the agents described above. [0093] Additionally, in compositions according to the present invention, the composition can further comprise a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b-cyclodextrin. When the composition comprises an additional therapeutic agent for the treatment of AML, the additional therapeutic agent can include one or more of brequinar sodium; a hypomethylating agent selected from the group consisting of azacytidine and decitabine; and all-frans-retinoic acid, in addition to the agents described above.

[0094] In methods according to the present invention, when at least one additional therapeutic agent for the treatment of AML is administered, the at least one additional therapeutic agent can be selected from the group consisting of: brequinar sodium; a hypomethylating agent selected from the group consisting of azacytidine and decitabine; and all-frans-retinoic acid.

[0095] In methods according to the present invention, the bisantrene can inhibit proliferation of leukemia stem/initiating cells (LSCs/LICs) to prevent repopulation of AML cells. In methods according to the present invention, the bisantrene can inhibit expression of immune checkpoint gene LILRB4. In methods according to the present invention, the bisantrene can sensitize AML cells to T-cell cytotoxicity.

[0096] Figure 1 is a graph showing two-year survival and residual disease status at transplant for AML patients.

[0097] Figure 2 shows Measurable Residual Disease in peripheral blood after the second cycle of chemotherapy and clinical outcomes. Shown are the rates of overall survival (Panel A) and the cumulative incidence of relapse in all patients (Panel B), in those without FLT3- ITD mutations (Panel C) and those with FLT3- ITD mutations (Panel D), and in those without DNMT3A mutations (Panel E) and those with DNMT3A mutations (Panel F) among patients who were found to have measurable residual disease (MRD-positive) or no measurable residual disease (MRD-negative) in peripheral-blood samples.

[0098] Figure 3 shows the association between pretransplant disease status and outcome for patients with acute myeloid leukemia (AML) after myeloablative hematopoietic cell transplantation (HCT). Estimates of (A) overall survival, (B) progression-free survival, (C) cumulative incidence of relapse, and (D) cumulative incidence of nonrelapse mortality (NRM) after myeloablative allogeneic HCT for adults with AML, shown individually for patients in measurable residual disease (MRD) - negative (n = 235) and MRD- positive (n = 76) morphologic remission as well as those with active AML (n = 48).

[0099] Figure 4 shows relapse among patients with non-DTA mutations.

[0100] Figure 5 shows the rate of relapse according to results of next-generation sequencing and multiparameter flow cytometry.

[0101] Figure 6 shows bisantrene monotherapy trial design to improve MRD(-) status before hematopoietic stem cell transplantation. (A) post-consolidation model; (B) second induction model.

[0102] As shown in Figure 6(A), another aspect of the invention is a method for treatment of AML comprising:

(1 ) performing high intensity induction therapy comprising administration of a therapeutically effective quantity of an anti-neoplastic agent for treatment of AML other than bisantrene to a patient to produce complete remission;

(3) determination of the presence or absence of MRD subsequent to step (2); (4) if MRD is found to be present in step (3), administration of a therapeutically effective quantity of bisantrene to convert the patient to MRD-negative status;

(5) if MRD is found to be absent in step (3), performance of a hematopoietic stem cell transplant leading to cure; and

(6) subsequent to the administration of the therapeutically effective quantity of bisantrene to convert the patient to MRD-negative status, performance of a hematopoietic stem cell transplant leading to cure.

[0103] As shown in Figure 6(B), yet another aspect of the invention is a method for treatment of AML comprising:

(2) determination of the presence or absence of MRD in the patient following step (1);

(3) if MRD is found to be present in step (2), administration of a therapeutically effective quantity of bisantrene;

(4) determination of the presence or absence of MRD in the patient following step (3);

(6) performance of a hematopoietic stem cell transplant leading to cure subsequent to step (5) or subsequent to step (4) if MRD is found to be absent in step (4) leading to cure.

[0104] In these methods based on Figures 6(A) and 6(B), the anti-neoplastic agent for treatment of AML other than bisantrene is as described above. [0105] Yet another aspect of the invention is a pharmaceutical composition formulated for the treatment of MRD comprising: (1) a therapeutically effective quantity of bisantrene or a derivative or analog of bisantrene; and (2) at least one pharmaceutically acceptable excipient.

[0106] Typically, the pharmaceutical composition comprises bisantrene.

[0107] Typically, the at least one pharmaceutically acceptable excipient is selected from the group consisting of:

(i) a liquid carrier;

(ii) an isotonic agent;

(iii) a wetting or emulsifying agent;

(iv) a preservative;

(v) a buffer;

(vi) an acidifying agent;

(vii) an antioxidant;

(viii) an alkalinizing agent;

(ix) a carrying agent;

(x) a chelating agent;

(xi) a coloring agent;

(xii) a complexing agent;

(xiii) a solvent;

(xiv) a suspending and/or viscosity-increasing agent;

(xv) an oil;

(xvi) a penetration enhancer;

(xvii) a polymer;

(xviii) a stiffening agent;

(xix) a protein;

(xx) a carbohydrate;

(xxi) a bulking agent; and (xxii) a lubricating agent. [0108] Other pharmaceutically acceptable carriers known in the art can be used.

[0109] In another alternative, the pharmaceutical composition can comprise a therapeutically effective quantity of at least one additional agent for the treatment of AML as described above.

[0110] Additionally, as described above, the composition can further comprise a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b-cyclodextrin.

[0111] In yet another alternative, the pharmaceutical composition can comprise a liposome. A liposomal formulation suitable for bisantrene or a derivative or analog thereof comprises small unilamellar or multilamellar liposomes of size range between 0.01 and 100 mM, and between about 50-95% liposome-entrapped bisantrene, composed of hydrogenated soy phosphatidylcholine, distearoyl phosphatidylglycerol, and cholesterol of natural or synthetic origin lipids, in aqueous solution which can be reconstituted from a lyophilized form to an injectable liposome suspension. The composition is prepared by reconstituting a lyophilized bisantrene/liposome composition to a liposome concentrate, then diluting the concentrate for parenteral administration for the treatment of AML.

ADVANTAGES OF THE INVENTION

[0112] The present invention provides a new paradigm for treating of acute myeloid leukemia (AML) focusing on the elimination of measurable residual disease (MRD) by the administration of bisantrene, an antineoplastic agent that has multiple mechanisms of action, including DNA intercalation, inhibition of topoisomerase, and activation of the immune system. Bisantrene is well tolerated and, in particular, lacks the cardiotoxicity that is characteristic of some other anthracene derivatives. Bisantrene can be used together with other therapeutic agents that are used for the treatment of AML.

[0113] Methods according to the present invention possess industrial applicability for the preparation of a medicament for the treatment of AML or for the use of the agents described herein for the treatment of AML, and compositions according to the present invention possess industrial applicability as pharmaceutical compositions for the treatment of AML.

[0114] Where methods are referred to, the methods of the present invention provide specific method steps that are more than general applications of laws of nature and require that those practicing the method steps employ steps other than those conventionally known in the art, in addition to the specific applications of laws of nature recited or implied in the claims, and thus confine the scope of the claims to the specific applications recited therein. In some contexts, these claims are directed to new ways of using an existing drug.

[0115] The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. As used herein the use of the term “comprising” as a transitional phrase in claims is intended to include therein the use of the transitional phrases “consisting essentially of” or “consisting of” if the narrower transitional phrases are not expressly excluded. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein.

[0116] In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent publications, are incorporated herein by reference.

Claims

What is claimed is:

1. A method of converting an AML patient, subsequent to initial treatment, from MRD(+) status to MRD(-) status, comprising the step of administering a therapeutically effective quantity of bisantrene to the patient.

2. The method of claim 1 wherein the bisantrene is administered for 7 days at a dosage from about 200 mg/m²/day to about 300 mg/m²/day.

3. The method of claim 2 wherein the bisantrene is administered for 7 days at a dosage from about 225 mg/m²/day to about 275 mg/m²/day.

4. The method of claim 3 wherein the bisantrene is administered for 7 days at a dosage of about 250 mg/m²/day.

5. The method of claim 1 wherein the bisantrene is administered for a period of 5 days, 6 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

6. The method of claim 1 wherein the bisantrene is administered intravenously.

7. The method of claim 6 wherein the bisantrene is administered centrally.

8. The method of claim 6 wherein the bisantrene is administered peripherally.

9. The method of claim 1 wherein the bisantrene is administered as a drug compound.

10. The method of claim 1 wherein the bisantrene is administered as a pharmaceutical composition, wherein the pharmaceutical composition includes at least one pharmaceutically acceptable carrier.

11. The method of claim 1 wherein the bisantrene is administered in a formulation comprising a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b- cyclodextrin.

12. The method of claim 11 wherein the dosage of bisantrene is about 15 mg/m² once every other day, 10 times.

13. The method of claim 1 wherein the bisantrene is administered in a liposome.

14. The method of claim 1 further comprising administration of at least one additional therapeutic agent for the treatment of AML.

15. The method of claim 14 wherein the at least one additional therapeutic agent for the treatment of AML is selected from the group consisting of:

(a) arsenic trioxide;

(b) daunorubicin hydrochloride;

(c) cyclophosphamide;

(d) cytarabine;

(e) glasdegib maleate;

(f) dexamethasone;

(g) doxorubicin hydrochloride;

(h) enasidenib mesylate;

(i) gemtuzumab ozogamicin;

(j) gilteritinib fumarate;

(k) idarubicin hydrochloride; (L) ivosidenib;

(m) midostaurin;

(n) mitoxantrone hydrochloride;

(o) thioguanine;

(p) venetoclax;

(q) vincristine sulfate;

(r) a combination of cytarabine, daunorubicin hydrochloride, and etoposide phosphate (ADE);

(s) an mRNA demethylase inhibitor;

(t) an activator of METTL3 or METTL14 selected from the group consisting of methylpiperidine-3-carboxylate hydrochloride and methylpiperazine-2- carboxylate;

(u) brequinar sodium;

(v) a hypomethylating agent selected from the group consisting of azacytidine and decitabine; and

(w) all-frans-retinoic acid.

16. The method of claim 15 wherein the at least one additional therapeutic agent for the treatment of AML is an mRNA demethylase inhibitor.

17. The method of claim 16 wherein the mRNA demethylase inhibitor is selected from the group consisting of rhein; meclofenamic acid; meclofenamic acid ethyl ester; a compound of Formula (F-lll)

(F-lll); a compound of Formula (F-IV)

(F-IV); which is an ethyl ester of the compound of Formula (F-lll); the R-enantiomer of 2- oxoglutarate; FB23, a derivative of meclofenamic acid; and FB23-2, a derivative of FB- 23.

18. The method of claim 17 wherein the mRNA demethylase inhibitor is

FB23-2.

19. The method of claim 1 wherein the bisantrene inhibits proliferation of leukemia stem/initiating cells (LSCs/LICs) to prevent repopulation of AML cells.

20. The method of claim 1 wherein the bisantrene inhibits expression of immune checkpoint gene LILRB4.

21. The method of claim 1 wherein the bisantrene sensitizes AML cells to T-cell cytotoxicity.

22. The method of claim 1 further comprising the step of determining MRD status in the patient.

23. The method of claim 22 wherein MRD status is determined prior to initiation of treatment.

24. The method of claim 22 wherein MRD status is determined during treatment as a marker of the progress of treatment.

25. The method of claim 22 wherein MRD status is determined by next- generation sequencing.

26. The method of claim 22 wherein MRD status is determined by multiparameter flow cytometry.

27. The method of claim 22 wherein MRD status is determined by a combination of both next-generation sequencing and multiparameter flow cytometry.

28. The method of claim 1 further comprising the step of detecting FLT3 mutations in the patient.

29. The method of claim 28 wherein the FLT3 mutations are internal tandem duplications.

30. The method of claim 28 wherein the FLT3 mutations are mutations in the tyrosine kinase domain.

31. The method of claim 1 further comprising the step of detecting DNMT3A mutations in the patient.

32. The method of claim 1 further comprising the step of detecting at least one mutation selected from the group consisting of a mutation in TET2, a mutation in ASXL1, and a mutation in NPM1 in the patient.

33. A method for treating AML comprising:

(a) performing high intensity induction therapy comprising administration of a therapeutically effective quantity of an anti-neoplastic agent for treatment of AML other than bisantrene to a patient to produce complete remission;

(b) consolidation of the AML resulting from the high intensity induction therapy of step (a); (c) determining the presence or absence of MRD subsequent to step

(b);

(d) if MRD is found to be present in step (c), administering a therapeutically effective quantity of bisantrene to convert the patient to MRD-negative status;

(e) if MRD is found to be absent in step (c), performing a hematopoietic stem cell transplant leading to cure; and

(f) subsequent to the administration of the therapeutically effective quantity of bisantrene to convert the patient to MRD-negative status, performing a hematopoietic stem cell transplant leading to cure.

34. The method of claim 33 wherein the at least one additional therapeutic agent for high intensity induction therapy is selected from the group consisting of:

(a) arsenic trioxide;

(b) daunorubicin hydrochloride;

(c) cyclophosphamide;

(d) cytarabine;

(e) glasdegib maleate;

(f) dexamethasone;

(g) doxorubicin hydrochloride;

(h) enasidenib mesylate;

(i) gemtuzumab ozogamicin;

(j) gilteritinib fumarate;

(k) idarubicin hydrochloride;

(L) ivosidenib;

(m) midostaurin;

(n) mitoxantrone hydrochloride;

(o) thioguanine;

(p) venetoclax;

(q) vincristine sulfate; (r) a combination of cytarabine, daunorubicin hydrochloride, and etoposide phosphate (ADE);

(s) an mRNA demethylase inhibitor;

(u) brequinar sodium;

(w) all-frans-retinoic acid.

35. The method of claim 33 wherein the bisantrene is administered in a formulation comprising a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b- cyclodextrin.

36. A method for treatment of AML comprising:

(a) performing high intensity induction therapy comprising administering a therapeutically effective quantity of an anti-neoplastic agent for treatment of AML other than bisantrene to a patient to produce complete remission;

(b) determining the presence or absence of MRD in the patient following step (a);

(c) if MRD is found to be present in step (b), administering a therapeutically effective quantity of bisantrene;

(d) determining the presence or absence of MRD in the patient following step (c);

(e) consolidation of the AML subsequent to step (b) if MRD is found to be absent in step (b) or subsequent to step (d) if MRD is found to be present in step (d); and (f) performing a hematopoietic stem cell transplant leading to cure subsequent to step (e) or subsequent to step (d) if MRD is found to be absent in step (d) leading to cure.

37. The method of claim 36 wherein the at least one additional therapeutic agent for high intensity induction therapy is selected from the group consisting of:

(a) arsenic trioxide;

(b) daunorubicin hydrochloride;

(c) cyclophosphamide;

(d) cytarabine;

(e) glasdegib maleate;

(f) dexamethasone;

(g) doxorubicin hydrochloride;

(h) enasidenib mesylate;

(i) gemtuzumab ozogamicin;

(j) gilteritinib fumarate;

(k) idarubicin hydrochloride;

(L) ivosidenib;

(m) midostaurin;

(n) mitoxantrone hydrochloride;

(o) thioguanine;

(p) venetoclax;

(q) vincristine sulfate;

(s) an mRNA demethylase inhibitor;

(u) brequinar sodium; (v) a hypomethylating agent selected from the group consisting of azacytidine and decitabine; and

(w) all-frans-retinoic acid.

38. The method of claim 36 wherein the bisantrene is administered in a formulation comprising a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b- cyclodextrin.

39. A pharmaceutical composition formulated for the treatment of MRD comprising:

(a) a therapeutically effective quantity of bisantrene; and

(b) at least one pharmaceutically acceptable excipient.

40. The pharmaceutical composition of claim 39 wherein the pharmaceutically acceptable excipient is selected from the group consisting of:

(i) a liquid carrier;

(ii) an isotonic agent;

(iii) a wetting or emulsifying agent;

(iv) a preservative;

(v) a buffer;

(vi) an acidifying agent;

(vii) an antioxidant;

(viii) an alkalinizing agent;

(ix) a carrying agent;

(x) a chelating agent;

(xi) a coloring agent;

(xii) a complexing agent;

(xiii) a solvent;

(xiv) a suspending and/or viscosity-increasing agent;

(xv) an oil; (xvi) a penetration enhancer;

(xvii) a polymer;

(xviii) a stiffening agent;

(xix) a protein;

(xx) a carbohydrate;

(xxi) a bulking agent; and (xxii) a lubricating agent.

41. The pharmaceutical composition of claim 39 wherein the composition further comprises a bioavailability enhancer selected from the group consisting of mPEG-b-PLA (methoxy polyethylene glycol-b-poly(D,L-lactide) micelles and b-cyclodextrin.

42. The pharmaceutical composition of claim 39 wherein the pharmaceutical composition comprises a therapeutically effective quantity of at least one additional agent for the treatment of AML.

43. The pharmaceutical composition of claim 42 wherein the at least one additional therapeutic agent for the treatment of AML is selected from the group consisting of:

(a) arsenic trioxide;

(b) daunorubicin hydrochloride;

(c) cyclophosphamide;

(d) cytarabine;

(e) glasdegib maleate;

(f) dexamethasone;

(g) doxorubicin hydrochloride;

(h) enasidenib mesylate;

(i) gemtuzumab ozogamicin;

(j) gilteritinib fumarate;

(k) idarubicin hydrochloride; (L) ivosidenib;

(m) midostaurin;

(n) mitoxantrone hydrochloride;

(o) thioguanine;

(p) venetoclax;

(q) vincristine sulfate;

(s) an mRNA demethylase inhibitor;

(u) brequinar sodium;

(w) all-frans-retinoic acid.

44. The pharmaceutical composition of claim 39 wherein the pharmaceutical composition comprises a liposome.