Methods for Treating and Ameliorating Cancer

Abstract

Methods for treating and ameliorating cancer or recurrence of cancer, such as acute myeloid leukemia (AML), are provided comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 (or revexinib) and a second drug, such as an ATP-competitive protein tyrosine kinase inhibitor (e.g., dasatinib). Methods for in vivo inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation are provided comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 and a second drug. Methods for in vivo inhibition of pre-leukemic stem cell (pre-LSC) transformation to leukemic stem cells (LSCs) are provided comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 and a second drug.

Description

[Technical field]

[0001] Related Applications This Patent Cooperation Treaty (PCT) international application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. (USSN) 63 / 144,378, filed February 1, 2021. The foregoing application is expressly incorporated by reference herein in its entirety and for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated by reference for all purposes.

[0002] STATEMENT REGARDING GOVERNMENT-SPONSORED RESEARCH This invention was made with government support under R01DK114468 and R01CA205944 awarded by the National Institutes of Health (NIH) and 80JSC020F0200 awarded by NASA. The Government has certain rights in this invention.

[0003] Technical Field The present invention generally relates to medicine and pharmacology. In an alternative embodiment, a method for treating and ameliorating cancer or relapse of cancer (e.g., acute myeloid leukemia (AML)) is provided, comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 (also known as rebeccinib) and a second drug (e.g., an ATP-competitive protein tyrosine kinase inhibitor (e.g., fedratinib or dasatinib)). In an alternative embodiment, a method for in vivo inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation is provided, comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 (rebeccinib) and a second drug. In an alternative embodiment, a method is provided for in vivo inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSCs), the method comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 (rebexinib) and a second drug. [Background technology]

[0004] background Accumulating evidence suggests that dormant self-renewing cancer stem cells (CSCs), especially in hematological malignancies (e.g., myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM)), promote treatment resistance to chemotherapeutic agents that target dividing cells, thus promoting relapse, which is the leading cause of cancer-related deaths. Relapse of these malignancies has been associated with splicing deregulation-induced clonal evolution that drives the expansion of CSCs (Jiang 2017). Summary of the Invention [Means for solving the problem]

[0005] Abstract In an alternative embodiment, (a) Rebeccinibe (also known as 17S-FD-895) [ka] or an enantiomer, stereoisomer, deuterated version, or salt thereof; and (b) at least one second drug; The present invention provides a pharmaceutical or therapeutic composition, formulation or therapeutic combination of drugs comprising:

[0006] In an alternative embodiment, - the treatment and amelioration of cancer, optionally wherein said cancer is leukemia, optionally wherein said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - Inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; - Inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSCs) in vivo; - in vivo inhibition of the spliceosome in AML or MPN; and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); 23. A method for the manufacture of a ... comprising: (a) Revexinib, also known as 17S-FD-895 [ka] or an enantiomer, stereoisomer, deuterated version, or salt thereof, or (b) the compound of (a), or revexinib or 17S-FD-895, and at least one second drug; administering to an individual in need thereof a formulation, pharmaceutical composition or therapeutic combination of drugs comprising A method is provided that includes:

[0007] In an alternative embodiment of the methods or pharmaceutical or therapeutic compositions, formulations or therapeutic combinations of drugs as provided herein, - a dose of 17S-FD-895 is administered once daily for 1-2 weeks, twice weekly for 2 weeks or about 1-2 weeks, followed by 2 weeks off or 2-4 weeks off, continuing for 2-week, 3-week, 4-week, 5-week or 6-week cycles, as needed for four 28-day or monthly cycles, or the pharmaceutical or therapeutic composition, formulation or therapeutic combination of drugs is formulated for administration of this dosage; - the at least one second drug comprises an ATP-competitive protein tyrosine kinase inhibitor, optionally wherein the ATP-competitive protein tyrosine kinase inhibitor is selected from the group consisting of dasatinib (or Sprycel TM Or Dasanix TM ) included; - the at least one second drug is a JAK2 (Janus kinase 2) inhibitor, optionally fedratinib (or INREBIC TM ), or fedratinib and at least one second drug, where optionally said fedratinib is administered at 60 mg / kg orally twice daily for 1-2 weeks or more as needed; - the at least one second drug comprises a chemotherapeutic agent, optionally wherein the chemotherapeutic agent is afatinib (or GILOTRIF TM ), afuresertib, alectinib, alisertib, alvocidib, amsacrine, amonafide, amuvatinib, axitinib, azacitidine, azathioprine, bafetinib, barasertib, bendamustine, bleomycin, bosutinib, bortezomib, busulfan, cabozantinib, camptothecin, canertinib, capecitabine, cabazitaxel, carboplatin, carmustine, cenisertib, ceritinib, chlorambucil, cisplatin, cladribine, Lofarabine, crenolanib, crizotinib, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dacomitinib, dactinomycin, danusertib, dasatinib, daunorubicin, decitabine, dinaciclib, docetaxel, dovitinib, doxorubicin, epirubicin, epitinib, eribulin mesylate, erlotinib, etirinotecan, etoposide, everolimus, exemestane, fedratinib (or INREBIC TM), floxuridine, fludarabine, fluorouracil, gefitinib, gemcitabine, hydroxyurea, ibrutinib, icotinib, idarubicin, ifosfamide, imatinib, ipatasertib, irinotecan, ixabepilone, lapatinib, lenalidomide, lestaurtinib, lomustine, lucitanib, masitinib, mechlorethamine, melphalan, mercaptopurine, methotrexate, midostaurin, mitomycin, mitoxantrone, mubritinib, nelarabine, neratinib, nilotinib, nintedanib, omacetaxine mepesuccinate, orantinib, oxaliplatin, paclitaxel, palbociclib, palifosfamide tris(palifosfamide tris), pazopanib, pelitinib, pemetrexed, pentostatin, plicamycin, ponatinib, poziotinib, pralatrexate, procarbazine, quizartinib, raltitrexed, regorafenib, ruxolitinib (or OPZELURA TM ), seliciclib, sorafenib (or NEXAVAR TM ), streptozocin, surufatinib, sunitinib (or SUTENT TM ), Tamoxifen (or NOLVADEX TM ), tandutinib, temozolomide, temsirolimus, teniposide, theliatinib, thioguanine, thiotepa, topotecan, uramustine, valrubicin, vandetanib, vemurafenib (or Zelborae TM ), vincristine (or ONCOVIN TM ), vinblastine (or VELBAN TM ), vinorelbine (or NAVELBINE TM ), and vindesine (or eldisine); - the at least one second drug comprises a hypomethylating agent (HMA), optionally wherein the HMA is azacitidine (or VIDAZA TM ) or decitabine (or DACOGEN TM ) including; - the at least one second drug comprises a second telomerase inhibitor, optionally wherein the telomerase inhibitor is selected from the group consisting of imetelstat, zidovudine (or azidothymidine (AZT)), stavudine (or ZERIT TM ), tenofovir or tenofovir disoproxil (or VIREAD TM ), didanosine (or VIDEX TM ), abacavir (ZIAGEN TM ), TMPI, telomestatin, RHPS4, BRACO-19, TMPyP4, tertomotide, ASTVAC-1, GX-301, UCPVax, UV-1, Vx-001, Vx-006, INO-1400, INVAC-1, ASTVAC-2, Telin (ab 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone), Vbx-011, Vbx-021, Vbx-026, INO-5401, KML-001, TK-005, ribovax, Vbx-016, ZI-HX, ZI-H04, and ZIH-03; - said formulation, pharmaceutical composition or therapeutic combination of drugs or the active agent or drug contained therein is, formulated in or contained in a liquid formulation (optionally sterile saline or water), spray, powder, aerosol, mist or any formulation for inhalation, pill, capsule, tablet, or gel tab, or equivalent); or is coated on or contained in a bead, powder, particle, or multi-layered bead or particle, optionally said bead, powder, particle or said multi-layered bead or particle being contained in a pill, capsule, tablet, or gel tab, or equivalent for oral delivery, optionally said pill, capsule, tablet, gel tab, or equivalent for oral delivery being a hard gelatin capsule or equivalent or containing hard gelatin or equivalent; or a drug delivery device or package, blister pack, clamshell, or tray, which contains a plurality of compartments spatially arranged on said drug delivery device or package, blister pack, clamshell, or tray to follow a dosage regimen; - the active agent used in the methods as provided herein, or the drug in the formulation, pharmaceutical composition, or therapeutic combination of drugs as provided herein, is administered at between about 10-500 mg / day, or between about 500-1 g per day, or at a dose of between about 100-600 mg per day or dosage, or at about 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg per day or dosage, optionally with a unit dose administered once per day (QD), or twice per day (BID), or three times per day (TID), or more, to an individual in need; - the active drug used in the methods as provided herein, or the drug in the formulation, pharmaceutical composition, or therapeutic combination of drugs as provided herein, is administered as or formulated with an inhaled or aerosol formulation (e.g., powder or mist or aerosol) and / or is formulated with or formulated as an oral, intramuscular (IM), subcutaneous (SC), intrathecal, or intravenous (IV) formulation, optionally with said inhaled (or aerosol) formulation and said oral, IV, SC, intrathecal, and / or IM formulation administered simultaneously or sequentially; - an active drug used in a method as provided herein, or a drug within a formulation, pharmaceutical composition or therapeutic combination of drugs as provided herein, is administered to an individual in need: optionally by inhalation using a drug delivery device, wherein said drug delivery device optionally comprises an inhalation device or inhaler or a nasal spray device, optionally said inhaler or nasal spray device is a hand-held inhaler or nasal spray device, optionally said inhaler or nasal spray device is a metered or metered dose inhaler or nasal spray device, or is used intravenously (IV) or intramuscularly (IM).

[0008] In an alternative embodiment, - the treatment and amelioration of cancer, optionally wherein said cancer is leukemia, optionally wherein said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - Inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; or - Inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSCs) in vivo - in vivo inhibition of the spliceosome in AML or MPN; and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); In accordance with the present invention, a pharmaceutical composition, formulation or therapeutic combination comprising 17S-FD-895 (or revexinib), or 17S-FD-895 and at least one second drug, for use in treating

[0009] In an alternative embodiment, - the treatment and amelioration of cancer, optionally wherein said cancer is leukemia, optionally wherein said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - Inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; or - Inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSCs) in vivo - in vivo inhibition of the spliceosome in AML or MPN; and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); In accordance with the present invention, there is provided a pharmaceutical composition, formulation or therapeutic combination comprising 17S-FD-895 (or revexinib), or 17S-FD-895 and at least one second drug, for use in the manufacture of a medicament or pharmaceutical composition for.

[0010] All publications, patents, and patent applications cited herein are expressly incorporated by reference in their entirety for all purposes.

[0011] Description of the drawings The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] The drawings presented herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

[0013] The drawings will now be described in detail. [Brief description of the drawings]

[0014] [Figure 1-1] Figure 1A-F illustrates that revexinib (17S-FD-895) significantly inhibited human CD34+Lin- (hCD34+) cell engraftment in the NSG-SGM3 mouse model transplanted with secondary AML patient 50261 (sAML50261), which secretes human stem cell factor, GM-CSF and IL-3.

[0015] FIG. 1A graphically depicts the percentage of engrafted human CD45+ (hCD45+) cells in peripheral blood (sAML37PB) of immunocompromised mice transplanted with secondary AML patient 37 after treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895).

[0016] FIG. 1B graphically depicts % CD45 positive (human CD45+ (hCD45+) cells in spleens (sAML37SP) in sAML-engrafted mice following treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895).

[0017] FIG. 1C graphically depicts % human CD45 positive cells (hCD45+) in bone marrow of secondary AML patient 37 engrafted mice (sAML37BM) following treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895).

[0018] [Figure 1-2] FIG. 1D graphically depicts % CD34 positive Lin negative cells (CD34+ (hCD34+ Lin-) in mice engrafted with secondary AML patient 37 peripheral blood (sAML37PB) after treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895).

[0019] FIG 1E shows the % CD34 positive Lineage negative cells (hCD34) in the spleens of secondary AML patient 37 transplanted mice (AML37SP) after treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895). + Lin - The graph shows the number of cells.

[0020] Figure IF graphically depicts % CD34 positive Lineage negative cells (hCD34+) in bone marrow of secondary AML Patient 37 engrafted mice (sAML37BM) following exposure to vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895), where all p-values ​​were calculated using unpaired T-tests.

[0021] [Diagram 2]FIG. 2 shows spleen weights (in mg) of secondary AML patient 37 transplanted (sAML37) NSG-SGM3 mice secreting human stem cell factor, GM-CSF, and IL-3 after treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895), graphically demonstrating that spleen size in the combination treatment group was significantly smaller after primary transplantation; all p values ​​were calculated using unpaired T-tests.

[0022] [Diagram 3] Figure 3 graphically illustrates the reduction in serially transplantable leukemia stem cells (LSCs) as shown by a significant reduction in spleen size in RAG2- / -gc- / - mice engrafted with human cells from mice treated with a combination of fedratinib and rebeccinib (rebcsinib) compared to groups treated with control, fedratinib, or rebeccinib (17S-FD-895). For this experiment, viable human CD45+ cells from secondary AML patient number 50261 (sAML 50261) engrafted mice were serially transplanted into RAG2- / -gc- / - mice lacking T cells, B cells, and NK cells after treatment with vehicle control, fedratinib, rebeccinib (17S-FD-895), or a combination of fedratinib and rebeccinib (17S-FD-895); all p values ​​were calculated using unpaired T-tests.

[0023] [Figure 4] Figures 4, 5 and 6A-C illustrate targeted inhibition of leukemic stem cells with rebeccinibb (17S-FD-895), where: rebeccinibb inhibits leukemic stem cells (LSCs) and reduces AML burden at doses that spare normal hematopoietic stem cells (HSCs) in vivo; LSCs are highly sensitive to splicing modulation with rebeccinibb in vivo; and splice isoform biomarkers include SF3B1, MCL1, CD44 and ADAR1.

[0024] FIG. 4 graphically depicts normal human cord blood hematopoietic stem cell (HSC) engraftment in RAG2− / −gc− / − mice based on flow cytometric quantification of the frequency of viable CD45 positive cells following administration of vehicle, 10 mg / kg revexinib, or 20 mg / kg revexinib.

[0025] [Diagram 5] FIG. 5 graphically depicts leukemia stem cell (LSC) survival based on frequency of viable CD34+CD38+ Lin factor progenitor cells (% baseline) following administration of vehicle or revexinib; p values ​​were calculated using an unpaired T-test.

[0026] [Figure 6] FIG. 6A graphically illustrates SF3B1 splicing factor intron retention by showing the SF3B1 intron 2 / total expression ratio following administration of vehicle or revexinib; p-values ​​were calculated using an unpaired T-test.

[0027] FIG. 6B graphically illustrates MCL1 levels by showing expression relative to the housekeeping gene, HPRT, after treatment with vehicle or revexinib; p-values ​​were calculated using an unpaired T-test.

[0028] FIG. 6C graphically depicts CD44-012 levels by showing expression relative to HPRT following administration of vehicle or revexinib; p values ​​were calculated using an unpaired T-test.

[0029] [Figure 7] Figures 7, 8 and 9 illustrate that lentiviral MAPT-GFP / RFP splicing reporter reveals intron retention following rebeccinib treatment and flow cytometry shows reduced ADAR p150 splice isoform expression following rebeccinib treatment.

[0030] FIG. 7 shows images of ADAR1 p150 positive live cells, with the inset showing a 100 μm scale.

[0031] [Figure 8] FIG. 8 graphically depicts the ratio of RFP (MFI) to GFP (MFI) following administration of DMSO vehicle control and 100 nM revexinib; p values ​​were calculated using an unpaired T-test.

[0032] [Figure 9] FIG. 9 graphically depicts ADAR1 P150 flow cytometry of the absolute number of viable ADAR1 P150 positive cells following administration of DMSO vehicle control and 100 nM revexinib; p values ​​were calculated using an unpaired T-test.

[0033] [Figure 10] Figures 10, 11, 12, 13, 14, 15, and 16 illustrate that inflammatory cytokine signaling drives ADAR1 p150 protein-induced splicing changes and that revexinib (17S-FD-895) inhibits high-risk myelofibrosis hematopoietic progenitor cell (HPC) survival at doses that inhibit ADAR1 activity by ADAR1 nanoluciferase reporter assay.

[0034] FIG. 10 illustrates diagrammatically the effect of APOBEC3C on pre-LSC cells and the effect of ADAR p150 on LSC cells.

[0035] [Figure 11] FIG. 11 shows relative mRNA expression normalized to human HPRT after administration of none (control) or various levels of rebeccinibb, graphically demonstrating that rebeccinibb inhibits ADAR1-p150 in a dose-dependent manner, as shown by qRT-PCR.

[0036] [Figure 12]FIG. 12 shows the amount of ADAR1 p150 mean fluorescence intensity (MFI) of live cells normalized to DMSO control after exposure to various amounts of rebexinib and graphically demonstrates that rebexinib inhibits AML patient number 193 (AML 193) progenitor cell ADAR1 p150 protein levels in a dose-dependent manner as shown by flow cytometry.

[0037] [Figure 13] FIG. 13 graphically illustrates that rebeccinibb inhibits ADAR1 nanoluciferase reporter activity at 1 μM in high-risk myelofibrosis progenitor cells using the ADAR1 nanoluciferase reporter and showing relative luciferase units normalized to cell viability following administration of DMSO control or 1 μM rebeccinibb.

[0038] [Figure 14] FIG. 14 graphically illustrates that rebeccinib inhibits myelofibrosis progenitor cell clonogenic (HPC) capacity to display CD34+CD38+ cells following administration of DMSO control or 1 μM rebeccinib.

[0039] [Figure 15] FIG. 15 graphically illustrates that rebeccinibb inhibits ADAR1 reporter activity in HPC progenitor cells at 1 μM rebeccinibb levels, showing ADAR1 p150 MFI of viable Lin negative, and CD34 positive, CD38 positive cells following treatment with DMSO control or 1 μM rebeccinibb.

[0040] [Figure 16] FIG. 16 shows RFP MFI of viable lineage CD34+CD38+ cells after administration of DMSO control or 1 μM rebexinib and graphically illustrates that rebexinib alters splicing reporter activity in high-risk myelofibrosis (hrMF) samples with high ADAR1 expression.

[0041] [Figure 17] FIG. 17 illustrates that IVIS imaging of ADAR1 nanoluciferase-GFP (ADAR1-nano-luc) reporter activity indicates normally aged human hematopoietic stem cell engraftment in RAG2− / −gc− / − mice transplanted intrathecally at birth and imaged 22 weeks post-transplant.

[0042] [Figure 18] FIG. 18 illustrates IVIS imaging of triple-negative breast cancer cells (MDA-MB-231) stably transduced with the ADAR1 nano-luc reporter injected into the ventricles of neonatal RAG2− / −g c− / − mice in the brain and spinal cord.

[0043] [Figure 19] FIG. 19 illustrates IVIS imaging of neonatal RAG2− / −g c− / − mice 6 weeks after intrahepatic implantation of triple-negative breast cancer cells (100,000 MDA-MB-231 cells / mouse) stably transduced with the ADAR1 nano-luc reporter.

[0044] [Figure 20] FIG. 20 illustrates a schematic of an exemplary method for making 17S-FD-895 (Rebexinib).

[0045] [Figure 21] FIG. 21A illustrates diagrammatically that the synthesis of 17S-FD-895 (1) occurred through the coupling of side chain 2 and core 3; and that the 11 sp3 stereocenters and stereochemistry of the three olefins of 1 arose from 12 precursors (inset) available on the kilogram scale.

[0046] FIG. 21B illustrates a schematic of the retro-analysis of a related macrolide, pladienolide B; shaded / colored highlighting indicates sourced components as shown in the gray (center) inset.

[0047] [Figure 22-1] Figures 22A-E, 23A-F and 24A-E graphically show flow cytometry analysis plots of ADAR1-nano-luc-GFP reporter expression (Y-axis) and CD44-APC (a breast cancer stem cell marker) on the X-axis in:

[0048] [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 22-4] Same as above. [Figure 22-5] Same as above.

[0049] [Figure 23-1] Figure 23A-F: Peripheral blood (PB), spleen (SP) and bone marrow (BM), and [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above. [Figure 23-5] Same as above. [Figure 23-6] Same as above.

[0050] [Figure 24-1] 24A-E Spinal cord and brain. In contrast to ventricular transplantation, engraftment is shown in the spinal cord, but not the brain, after intrahepatic transplantation. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 24-5] Same as above.

[0051] [Figure 25-1]25A-B graphically depict stromal co-culture assays in sAML (mutated splicing factors vs. non-mutated) and high-risk myelofibrosis (MF) vs. age-matched normal bone marrow (a-NBM) samples treated with 17S-FD-895.

[0052] FIG. 25A graphically depicts results showing a significant reduction in sAML LSC (n=3 unique samples (including splicing factor mutated and non-mutated samples)) survival (upper panel) and self-renewal (lower panel) following treatment with revexinib compared to a-NBM (n=4).

[0053] [Figure 25-2] Figure 25B shows the dose response of ABM vs. MF vs. sAML, and graphically illustrates the results of a-NBM vs. sAML vs. sAML non-mutated, showing a significant reduction in progenitor cell survival (upper panel) and self-renewal (lower panel) of high-risk MF pre-cancer after treatment with Revexinib compared to a-NBM (n=4). Grouped sAML LSC (n=9) is shown for reference. Statistical analysis was performed using one-way ANOVA.

[0054] [Figure 26] Figures 26A-B graphically depict in vitro stromal co-culture and protein expression assays in lentiviral ADAR1 reporter-transduced myelofibrosis (MF) samples treated with revexinib, where intracellular flow cytometry-based quantification of ADAR1 p150 protein expression (Figure 26A) and STAT3 phosphorylation (Figure 26B) are shown as represented by mean fluorescence intensity (MFI) within the HPC population; statistical analysis was performed using two-tailed pairwise t-tests; error bars represent the mean ± SEM.

[0055] [Figure 27-1] 27A-D graphically depict ADAR1 expression and activity, and pro-survival transcript expression in AML cells treated with revexinib in vitro.

[0056] FIG. 27A graphically depicts total ADAR1 mRNA expression in lentivirus-shCtrl- or shADAR1-transduced AML-193 cells treated with 17S-FD-895 or DMSO control.

[0057] [Figure 27-2] FIG. 27B graphically depicts RNA editing activity against the endogenous LSC-associated transcript AZIN1, as quantified by RNA editing site-specific qPCR (RESSqPCR), in AML-193 cells treated with 17S-FD-895 or DMSO control for 4 hours (left panel) or 24 hours (right panel).

[0058] [Figure 27-3] FIG. 27C graphically depicts splice isoform-specific qPCR demonstrating reduced MCL1-L expression following ADAR1 knockdown and / or 17S treatment for 4 hours (left panel) or 24 hours (right panel).

[0059] [Figure 27-4] FIG. 27D graphically depicts splice isoform-specific qPCR showing reduced CD44v3 expression following ADAR1 knockdown and / or 17S treatment for 4 hours; where:

[0060] Statistical analysis was performed using unpaired two-tailed Student's t-test (n=4 replicates / condition).

[0061] [Figure 28-1] Figures 28A-E illustrate 17S-FD-895 treatment in lentiviral splicing reporter assays, in which KG1a or MOLM13 human adult leukemia cells were stably transduced with the pCDH-EF1 a-lRES-Puro MAPT splicing reporter lentiviral vector.

[0062] 28A-B graphically depict intron retention (FIG. 28A) and viability (FIG. 28B) of KG-1a cells treated with 17S-FD-895 for 24 hours.

[0063] [Figure 28-2] Figures 28C-E graphically show data from a study in which MOLM13-MAPT cells were transplanted into irradiated NSG-SGM mice followed by 24-hour treatment with vehicle (n=3) and 17S-FD-895 (10 or 20 mg / kg; n=2, n=3), and the ratios of RFPIGFP median fluorescence intensity (MF / ) of viable CD45+ cells in bone marrow (Figure 28C), spleen (Figure 28D), and peripheral blood (Figure 28E) were quantified by flow cytometry, where p<0.05 by two-tailed Student's t-test.

[0064] Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Detailed Description In an alternative embodiment, methods are provided for treating and ameliorating cancer, e.g., leukemia (e.g., acute myeloid leukemia (AML)), comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 (also referred to as rebeccinib), and optionally a second drug (e.g., an ATP-competitive protein tyrosine kinase inhibitor (e.g., fedratinib or dasatinib)). In an alternative embodiment, methods are provided for the in vivo inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation, comprising administering to an individual in need thereof a pharmaceutical composition comprising 17S-FD-895 (rebeccinib), or 17S-FD-895 and a second drug. In an alternative embodiment, a method is provided for in vivo inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSCs), the method comprising administering to an individual in need thereof 17S-FD-895 (rebexinib), or a pharmaceutical composition comprising 17S-FD-895 and a second drug.

[0066] We have clearly demonstrated that 17S-FD-895 (Revexinib) significantly reduces the frequency of self-renewing CSCs in MPN, MDS, and both adult and pediatric AML-stromal co-cultures, as well as in humanized AML mouse models, at doses that preserve normal hematopoietic stem cell (HSC; CD34+Lin-) survival and differentiation into mature progeny. Functionally, 17S-FD-895 and related compounds reduce the regenerative potential of malignancies both in vitro and in vivo by impairing CSC self-renewal capacity while sparing normal human hematopoietic stem and progenitor cells (HSPCs). Thus, 17S-FD-895 exhibits a favorable therapeutic index and has potential utility in preventing disease recurrence in human hematologic and other malignancies.

[0067] Splicing modulators (e.g., E7107, H3B-8800, and 17S-FD-895 (Revexinib)) have efficacy in preclinical models of leukemia, but only 17S-FD-895 was stable, safe, and tolerated in preclinical models. One chemically distinct splicing modulator, E7107, entered clinical trials for solid tumors (Hong 2014), but two of 26 patients developed reversible optic neuritis associated with compound instability (Leon 2017), resulting in early discontinuation of the trial. Subsequent analysis suggested that other toxicities were related to metabolic degradation of E7107 to seco acids. In contrast, candidate 17S-FD-895 underwent metabolic degradation, but without toxic consequences. 17S-FD-895 offers the potential to evaluate splicing modulators in a clinical setting without the legal liability associated with compound instability and suboptimal pharmacology. Furthermore, E7107 has shown limited efficacy in clinical trial results (Hong, 2014), whereas there is a significant reduction in AML CSC survival and self-renewal following treatment with 17S-FD-895 in preclinical models.

[0068] At the molecular level, 17S-FD-895 regulates the splicing of several BCL2 pro-survival family members, including BCL2, MCL1, and BCL-XL (BCL2L1). This provides a new avenue to regulate this class of molecules, some of which are targets of other clinical therapeutics, such as venetoclax. BCL2L1 splice isoform switching contributes to LSC generation (Goff et al., 2013) and is particularly relevant to the unique mechanism of action (MOA) of 17S-FD-895 in sAML, because its transcripts showed resistance to E7107, another splicing modulator agent that previously entered clinical trials (Aird et al., Nature Communications 2019). Furthermore, we previously showed that sAML CSCs upregulate the splicing regulator gene ACIN1 (Crews et al., 2016). The gene regulates the generation of BCL2L1 (BCL-XL) at the exon junction complex (EJC) (Michelle et al., 2012). Moreover, MCL1L is overexpressed in high-risk MPNs and MDS, sAML, pediatric AML, and multiple myeloma. Furthermore, IL-6ST, ADAR1p150, and STAT3beta splice isoforms are overexpressed during MDS and MPN progression to AML. Thus, hematological malignancies may be particularly sensitive to splicing modulator treatment with 17S-FD-895 due to the intrinsic activation of these pro-survival and pro-self-renewal isoforms in CSCs and their subsequent regulation by 17S-FD-895. Quantification of CSC-specific and splicing modulator-responsive transcripts facilitates rapid screening of the in vivo sensitivity of different tissues to splicing modulation and identification of doses that show minimal effects on normal tissues compared to malignant cell types.

[0069] A phase 1 clinical trial in patients with MDS, AML and CMML was conducted with another chemically distinct splicing modulator, H3B-8800. H3B-8800 is a splicing factor mutation-dependent splicing modulator factor. The most common mutations observed were in RNA splicing factors SF3B1, U2AF1, SRSF2 (88% of patients). Furthermore, neither CR nor PR was observed in the trial, indicating a lack of efficacy associated with this splicing modulator. In contrast, through extensive pharmacological chemical and toxicological studies, we identified 17S-FD-895 as a stable, well-tolerated, spliceosome mutation-independent FD-895 analog (Kumar 2016).

[0070] Clinical feasibility for 17S-FD-895 (Revexinib) is based on extensive in vitro and in vivo preclinical studies demonstrating stability, safety, tolerability and human CSC targeting efficacy in humanized models of AML. Specifically, 17S-FD-895 has a favorable safety profile, with no ocular toxicity noted in toxicity studies in rats, rabbits and monkeys as determined by a certified board-certified ophthalmologist. Compared to other splicing modulators, 17S-FD-895 has favorable potency and therapeutic index in that it reduces disease regeneration potential in vitro and in vivo by impairing CSC self-renewal capacity while sparing normal human HSPCs. Finally, 17S-FD-895 has pharmacologic (PK / PD) properties with favorable bioavailability and stability that allow for a clinically tractable formulation and a twice-weekly intravenous dosing regimen that eliminates human CSCs. Sensitive and selective methods to predict and monitor response to splicing modulator treatment with 17S-FD-895 are essential for the future clinical development of these agents.

[0071] Although the present invention is not limited by any particular mechanism of action, 17S-FD-895 (Revexinib) inhibits transcripts that bind to SF3B1 and other components of the spliceosome binding pocket (including SF3B3 and PHF5A), which disrupts the production of important CSC survival transcripts, thereby inducing effective elimination of CSCs in AML samples regardless of splicing factor mutation status. This may broaden the scope of activity against other malignancies with splicing deregulation, in contrast to another splicing modulator factor, H3B-8800, which depends on splicing factor mutations such as SRSF2, in terms of altering its RNA binding preference and efficacy within the spliceosome (https: / / ashpublications.org / blood / article / 134 / Supplement_1 / 673 / 426543 / Results-of-a-Clinical-Trial-of-H3B-8800-a-Splice).

[0072] Splice isoform-specific molecular analyses characterizing the effects of 17S-FD-895 in AML models clearly show that, mechanistically, this compound potently perturbs the spliceosome, inducing quantifiable and reproducible intron retention and exon skipping events detectable in disease-associated (MCL1, CD44) and spliceosome-associated biomarkers (SF3B family). Functionally, we tested the relative in vitro sensitivity of primary patient-derived sAML CSCs to 17S-FD-895 compared to normal age-matched hematopoietic stem and progenitor cells (HSPCs) and determined the effect of splicing modulation on sAML CSC survival and self-renewal capacity. We also tested the in vitro effect of 17S-FD-895 on the hematopoietic differentiation potential of normal human HSPCs by comparing the in vitro effect of 17S-FD-895 on normal immune cell survival and development.

[0073] Biomarkers were developed and validated to monitor the response to splicing modulation in human and rat cells. Transcripts selected for species-specific primer design included MCL1, BCL-XL, CD44, PTK2B, DNAJB1, and SF3B families. Together, these biomarkers are diagnostic tools to predict and monitor the response to splicing modulator treatment.

[0074] Preparation of 17S-FD-895 17S-FD-895 (Revexinib) can be made using any protocol known in the art, for example, as described in Chan et al., Cell Reports Physical Science 1: (see FIG. 21) and as described in FIGS. 22A-B.

[0075] FIG. 21A illustrates diagrammatically that the synthesis of 17S-FD-895 (1) occurs through the coupling of side chain 2 and core 3. The 11 sp3 stereocenters and stereochemistry of the three olefins of 1 arose from 12 precursors (inset) available on the kilogram scale. The key steps used to prepare each building block are noted.

[0076] Figure 21B shows the results of core 5a from Ghosh and Anderson. 24 By and from Core 5b Kotake 28 FIG. 1 shows a schematic diagram of a retrospective analysis of a related macrolide, pladienolide B, as developed by Friedrichs et al. The shaded / colored highlighting indicates the sourced components, as shown in the grey (center) inset.

[0077] Formulations and Pharmaceutical Compositions In alternative embodiments, there are provided pharmaceutical formulations or compositions comprising drugs, and therapeutic combinations of drugs, and formulations, and liposomes for carrying out the methods and uses as provided herein for: For example, to treat or ameliorate cancer (wherein optionally the cancer is a leukemia, and optionally the cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); or for the in vivo inhibition of myeloproliferative neoplasms (MPN) or AML stem cell proliferation; or for the in vivo inhibition of pre-leukemic stem cell (pre-LSC) transformation to leukemic stem cells (LSCs), or for the in vivo inhibition of the spliceosome in AML or MPN, and / or for the in vivo inhibition of transcripts that bind to components of the spliceosome binding pocket or that bind to adenosine deaminase acting on RNA-1 (ADAR1).

[0078] In alternative embodiments, the formulations or pharmaceutical compositions used to carry out the methods and uses as provided herein can be administered parenterally, topically, orally, or by local administration (e.g., by aerosol or transdermally), or by intravitreal injection.The formulations and pharmaceutical compositions (including therapeutic drug combinations) can be formulated in any manner and administered in various unit dosage forms depending on the condition or disease and the extent of the disease, the overall medical condition of each patient, the resulting preferred method of administration, etc.Details regarding formulation and administration techniques are fully described in the scientific and patent literature.See, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co., Easton PA ("Remington's").

[0079] For example, in alternative embodiments, the compositions used to carry out the methods and uses as provided herein are formulated in buffer, saline, powder, emulsion, vesicle, liposome, nanoparticle, nanolipoparticle, etc. In alternative embodiments, the compositions can be formulated in any manner and applied in various concentrations and forms depending on the desired in vivo, in vitro, or ex vivo conditions, the desired in vivo, in vitro, or ex vivo method of administration, etc. Details regarding in vivo, in vitro, or ex vivo formulations and administration techniques are fully described in the scientific and patent literature. The formulations and / or carriers used to carry out the methods or uses as provided herein can be in the form of tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro, or ex vivo application.

[0080] In alternative embodiments, the formulations and pharmaceutical compositions used to carry out the methods and uses as provided herein may include solutions of the compositions (e.g., any active agent used in the methods provided herein) disposed or dissolved in a pharma- ceutically acceptable carrier, for example, acceptable vehicles and solvents that may be used include water and Ringer's solution, isotonic sodium chloride. In addition, sterile, fixed oils may be used as solvents or suspending media. For this purpose, any fixed oil may be used, including synthetic mono- or diglycerides, or fatty acids (e.g., oleic acid). In one embodiment, the solutions and formulations used to carry out the methods and uses as provided herein may be sterile and manufactured to be substantially free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well-known sterilization techniques.

[0081] The solutions and formulations used to practice the methods and uses as provided herein may contain auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of active agents in these formulations may vary widely and may be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected, in vivo, in vitro or ex vivo, and the desired results.

[0082] The compositions and preparations used for carrying out the methods and uses as provided herein can be delivered by using liposomes.By using liposomes, they can focus the delivery of active agents to target cells in in vivo, in vitro or ex vivo applications, especially when the liposome surface carries ligands specific to target cells (e.g., damaged or diseased nerve cells or CNS tissues) or is otherwise preferentially directed to specific tissue or organ types.

[0083] Nanoparticles, Nanolipoparticles and Liposomes Also provided are membranes of nanoparticles, nanolipoparticles, vesicles and liposomes comprising compounds used to deliver, e.g., drugs, for carrying out the methods and uses as provided herein, e.g., for expressing compositions used to carry out the methods as provided herein, for: For example, it may be used to treat or ameliorate cancer (wherein optionally the cancer is a leukemia, and optionally the cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); or for the in vivo inhibition of myeloproliferative neoplasms (MPN) or AML stem cell proliferation; or for the in vivo inhibition of pre-leukemic stem cell (pre-LSC) transformation to leukemic stem cells (LSCs), or for the in vivo inhibition of the spliceosome in AML or MPN, and / or for the in vivo inhibition of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1). In alternative embodiments, the compositions are designed to target specific molecules (including biological molecules such as polypeptides (including cell surface polypeptides)) for targeting a desired cell type or organ, such as, for example, neural cells or the CNS.

[0084] Multilamellar liposomes containing the compounds used to practice the methods and uses as provided herein are provided, for example as described in Park et al., U.S. Patent Publication No. 20070082042. The multilamellar liposomes may be prepared to entrap the compositions used to practice the methods and uses as provided herein using a mixture of oil phase components including squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithin with particle sizes of about 200-5000 nm.

[0085] Liposomes may be made using any method, as described, for example, in Park et al., U.S. Patent Publication No. 20070042031, including a method of generating liposomes by entrapping an active agent (e.g., a drug combination as described herein, or a nucleic acid encoding ADAR1, or an ADAR1 polypeptide), comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution and the organic lipid solution in a first mixing region to generate a liposome solution (wherein the organic lipid solution mixes with the aqueous solution to substantially instantly generate liposomes encapsulating the active agent); and immediately thereafter, mixing the liposome solution with a buffer solution to generate a diluted liposome solution.

[0086] In one embodiment, the liposome compositions used to practice the methods and uses as provided herein comprise substituted ammonium and / or polyanions, e.g., for targeted delivery of a compound (e.g., a drug or drug combination as provided herein) to a desired cell type (e.g., cancer cells), e.g., as described in U.S. Patent Publication No. 20070110798.

[0087] For example, nanoparticles are provided that contain a compound (e.g., a drug or drug combination as provided herein) in the form of an active agent-containing nanoparticle (e.g., secondary nanoparticle) as described in U.S. Patent Publication No. 20070077286. In one embodiment, nanoparticles are provided that contain a fat-soluble active agent or a water-soluble active agent dissolved in fat to act with a divalent or trivalent metal salt.

[0088] In one embodiment, solid lipid suspensions can be used to formulate and deliver compositions used to practice the methods and uses as provided herein to mammalian cells, e.g., to the CNS in vivo, e.g., as described in U.S. Patent Publication No. 20050136121.

[0089] Cell delivery and vehicle delivery In alternative embodiments, any delivery vehicle can be used to carry out the method or use as provided herein, for example, to deliver the composition (e.g., the drug or drug combination as provided herein) to an individual in need in vivo.For example, a delivery vehicle comprising a polycation, a cationic polyol and / or a cationic peptide (e.g., a polyethyleneimine derivative) can be used, for example, as described in, for example, US Patent Publication No. 20060083737.

[0090] In one embodiment, the dried polypeptide-surfactant complex is used to formulate compositions used to practice methods as provided herein, e.g., as described in U.S. Patent Publication No. 20040151766.

[0091] In one embodiment, the composition used to carry out the method and use as provided herein can be applied to cells using a vehicle with cell membrane permeable peptide conjugate, for example, as described in U.S. Patent No. 7,306,783; U.S. Patent No. 6,589,503.In one aspect, the composition to be delivered is conjugated to cell membrane permeable peptide.In one embodiment, the composition to be delivered and / or delivery vehicle is conjugated to transport mediating peptide, for example, as described in U.S. Patent No. 5,846,743, which describes a transport mediating peptide that is highly basic and binds to poly-phosphoinositides.

[0092] In alternative embodiments, the drugs or drug combinations as described herein are formulated in lipid formulations or liposomes and delivered in vivo using methods as provided herein, e.g., injected intramuscularly (IM), e.g., using formulations and methods as described in U.S. Patent Application No. US 20210046173 A1; where optionally the drug is formulated in liposomes comprising cholesterol and DSPC, or PEG-lipids, or PEG-modified lipids, or LNPs, or a mixture of ionizable cationic lipids; or a non-cationic lipid comprising a mixture of (13Z,16Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, cholesterol, DSPC, and PEG-2000 DMG, or lipid nanoparticles (LNPs), or nanoliposomes. In alternative embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), or the PEG-lipid is PEG linked to dimyristoylglycerol (PEG-DMG). In alternative embodiments, the LNP comprises 20-99.8 mol% ionizable cationic lipid, 0.1-65 mol% non-cationic lipid, and 0.1-20 mol% PEG-lipid.In an alternative embodiment, the LNP comprises an ionizable cationic lipid selected from the group consisting of (2S)-1-({6-[(3))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine; or a pharma- ceutically acceptable salt thereof, or a stereoisomer of any of the foregoing. In an alternative embodiment, the PEG-modified lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In an alternative embodiment, the ionizable cationic lipid comprises 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine. In one embodiment, the lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine or N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, each of which is described in PCT / US2011 / 052328, the entire contents of which are incorporated by reference.In some embodiments, non-cationic lipids of the present disclosure include: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DM PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphentanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or a mixture thereof.

[0093] Administration The pharmaceutical compositions, drug combinations and formulations used to practice the methods and uses as provided herein may be administered for prophylactic and / or therapeutic treatment, e.g., to treat or ameliorate cancer, where optionally said cancer is leukemia, where optionally said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); or may be used for in vivo inhibition of myeloproliferative neoplasms (MPN) or AML stem cell proliferation; or may be used for in vivo inhibition of pre-leukemic stem cell (pre-LSC) transformation to leukemic stem cells (LSCs), or may be used for in vivo inhibition of the spliceosome in AML or MPN, and / or may bind to components of the spliceosome binding pocket or inhibit ADAR1 (adenosine deaminase acting on and the pharmaceutical compositions, drug combinations and formulations used to practice the methods and uses as provided herein may be administered to an individual in need thereof in an amount sufficient to treat, ameliorate, protect against, reverse or reduce the severity or duration of said cancer.

[0094] The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." Effective administration schedules and amounts, i.e., "dosing regimens," for this use will depend on a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the patient's general health status, the patient's physical condition, age, etc. In calculating a patient's dosing regimen, the mode of administration will also be taken into consideration.

[0095] The pharmaceutical compositions, drug combinations and preparations used to carry out the methods and uses as provided herein can be administered as a single dose or in multiple doses, if required.In alternative embodiments, these doses are administered intravitreally, orally, IM, IV, or intrathecally.In alternative embodiments, the vector is administered as a formulation or pharmaceutical preparation, for example, the drug is contained in nanoparticles, particles, micelles, or liposomes or lipoplexes, polymersomes, polyplexes, or dendrimers.In alternative embodiments, these doses are administered once a day, once a week, or any variation thereof, if required, to maintain the desired in vivo expression level of the drug, which can be monitored by evaluating therapeutic effect, for example, to treat, improve, protect against, improve or reduce the severity or duration of cancer. The above dosage regimens also take into account pharmacokinetic parameters (i.e., rate of absorption, bioavailability, metabolism, clearance, etc. of the active agent) that are well known in the art (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; latest edition of Remington's, supra). The state of the art allows clinicians to determine the dosage regimen of each individual patient, active agent and disease or condition to be treated.Guidelines provided for similar compositions used as medicines can be used as guidance to determine the dosage regimen.That is, the dosage schedule and equivalent dose level administered to carry out the method as provided herein are accurate and appropriate.

[0096] Single or multiple administrations of the formulation, therapeutic drug combination may be given according to the dosage and frequency as required and tolerated by the patient. The formulation should provide a sufficient amount of active agent to effectively treat, prevent or ameliorate the condition, disease or symptoms as described herein. For example, alternative exemplary pharmaceutical formulations for oral administration of the compositions used to practice the methods as provided herein are in daily amounts of between about 0.1-0.5 to about 20, 50, 100 or 1000 or more μg per kg of body weight per day. In alternative embodiments, dosages are used from about 1 mg to about 4 mg per kg of body weight per patient per day. In contrast to oral administration, lower dosages may be used for administration into the bloodstream, into a body cavity, or into the lumen of an organ. Substantially higher dosages may be used for topical or oral administration, or administration by powder, spray or inhalation. Actual methods for preparing formulations suitable for parenteral or non-parenteral administration will be known or apparent to those skilled in the art and are described in detail in such publications as Remington's, supra.

[0097] The methods as provided herein may further include co-administration with other drugs or pharmaceuticals, e.g., compositions for treating any neurological or neuromuscular disease, condition, infection or injury, including associated inflammatory and autoimmune diseases and conditions, etc. For example, the methods and / or compositions and formulations as provided herein may be co-formulated and / or co-administered with fluids, antibiotics, cytokines, immunomodulatory agents, anti-inflammatory agents, pain relieving compounds, complement activators (e.g., peptides or proteins comprising a collagen-like or fibronectin-like domain (e.g., ficolins), carbohydrate binding domains, etc., and combinations thereof).

[0098] Manufacturing Products and Kits Articles of manufacture and kits are provided for use in practicing the methods as provided herein; optionally the articles of manufacture and kits may further include instructions for practicing the methods as provided herein.

[0099] Any of the above aspects and embodiments may be combined with any other aspect or embodiment as disclosed herein in the Summary, Drawings and / or Detailed Description sections. As used in this specification and the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0100] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to include and cover both "or" and "and."

[0101] Unless specifically stated or clear from the context, the term "about" as used herein is understood to mean within the normal tolerance in the art, for example, within 2 standard deviations of the mean, and about (use of the term "about") can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0102] Unless specifically stated or clear from the context, as used herein, the terms "substantially all," "substantially most of," "substantially all of," or "majority of" include at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the referenced amount of a composition.

[0103] Each patent, patent application, publication and document referenced herein thus far is incorporated by reference in its entirety. The citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is related to prior art, nor is it an admission that it constitutes any admission as to the contents or dates of these publications or documents. The incorporation of these documents by reference should not, in and of itself, be construed as a claim or admission that any portion of the contents of any document is deemed essential material to satisfy the legal disclosure requirements of any country or region for patent applications. Nevertheless, the right is reserved to rely on any of such documents, where appropriate, to provide material deemed essential to the claimed subject matter by an examining authority or court.

[0104] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the present invention has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, but that these modifications and improvements are within the scope and spirit of the invention. The invention as suitably illustrated herein may be practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms. Thus, the terms and expressions used are used as terms of description, but not as terms of limitation, and do not exclude equivalents of the features shown and described, or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. The embodiments of the invention are set forth in the following claims.

[0105] The present invention will be further described with reference to the examples set forth herein; however, it should be understood that the present invention is not limited to such examples. EXAMPLES

[0106] Working Example Unless otherwise stated in the examples, all recombinant DNA techniques are performed according to standard protocols, for example as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY, and of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, volumes 1 and 2. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, NY, Brown (1998) Molecular Biology LabFax, 2nd Edition, Academic Press (UK), volumes I and II. Standard materials and methods for polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al. (2000) PCR - Basics: From Background to Bench, 1st Edition, Springer Verlag, Germany.

[0107] Example 1: Exemplary methods of using 17S-FD-895 to reduce self-renewal of AML CSCs and for targeted CSC eradication This example clearly demonstrates that the methods and compositions as provided herein are effective in vivo for reducing the level of self-renewal or occurrence of AML CSCs and for targeted CSC eradication.

[0108] We have demonstrated that 17S-FD-895 (also called revexinib) significantly reduces the frequency of self-renewal of AML CSCs in stromal co-cultures and humanized AML mouse models at doses that preserve normal hematopoietic stem cell (HSC; CD34+CD90+Lin-) survival and differentiation into mature progeny. In AML CSCs, these effects are achieved through on-target splicing modulator effects, including MCL1 and splicing factor gene products (e.g., SF3B3, which forms part of the splicing modulator binding pocket).

[0109] We also developed and validated sensitive splice isoform biomarkers to detect SF3B family members that are easily quantifiable in qRT-PCR assays. Response to splicing modulator treatment can also be monitored in live cells using a lentiviral fluorescent splicing reporter, a novel tool compatible with use in clinical samples.

[0110] Splice isoform-specific molecular analyses characterizing the effects of 17S-FD-895 in AML models clearly show that, mechanistically, this compound potently perturbs the spliceosome, inducing quantifiable and reproducible intron retention and exon skipping events detectable in disease-associated (MCL1, CD44) and spliceosome-associated biomarkers (SF3B family), in vitro splicing modulator treatment and species-specific splice biomarker validation in human and rat cell lines.

[0111] Test system: In vitro cell line assays under treatment with 17S-FD-895 (1 nM-10 μM range). For in vitro treatment, human leukemia cell lines (KG-1a, MOLM-13, and HL-60) and rat leukemia line (RBL-1) were treated with 17S-FD-895 for 4 hours in standard cell culture medium for each line. Cells were harvested for RNA extraction and treated with splice isoform-specific primers designed to recognize intron retention or exon skipping events in species-specific transcripts of SF3B family members, MCL-1 and other sAML-specific splice variants for qRT-PCR.

[0112] Test Article: 17S-FD-895 (research and pre-pilot batches #IA01f and IK01f).

[0113] Control article: <0.5% DMSO.

[0114] Test and control article preparation: Test articles were thawed at room temperature from stock aliquots stored in glass vials at -80°C at a concentration of 1 mg / mL in 100% DMSO. Once thawed, test articles were diluted directly into tissue culture medium for final treatment at a concentration of 1 μM for single dose studies or in the range of 100 nM to 10 μM for dose response studies. For vehicle controls, 100% DMSO was diluted directly into tissue culture medium at the same dilution as the test article solutions.

[0115] In-Life Monitoring: Assay measurements included splice isoform-specific, species-specific qRT-PCR.

[0116] The alternative splicing modulator, 17S-FD-895, and related compounds show disease-modifying on-target efficacy against cancer stem cells in diverse human cancer models. We have shown that spliceosome deregulation in cancer stem cells (CSCs) renders them highly vulnerable to a novel splicing modulator, 17S-FD-895, which targets SF3B1 of the spliceosome [Kotake 2007] [Crews 2016]. Splicing modulation with 17S-FD-895 significantly reduces secondary acute myeloid leukemia (sAML) CSC burden in vivo, along with reduced CSC survival and self-renewal. Importantly, splicing modulation with 17S-FD-895 spares normal human hematopoietic progenitors in vivo, and 17S-FD-895 eradicates human CSCs regardless of mutational status. Thus, 17S-FD-895 fulfills an urgent unmet medical need for developing splicing modulator therapeutics to eradicate CSCs, thereby preventing AML treatment resistance and relapse.

[0117] The present invention is not limited by a specific mechanism of action, but mechanistically, 17S-FD-895 binds in the spliceosome adjacent to SF3B1, SF3B3, and PHF5A. Treatment with 17S-FD-895 increases intron retention and exon skipping, thereby reducing CSC survival and self-renewal. In cells derived from human age-related malignancies, including AML and multiple myeloma, these effects are accompanied by on-target splicing modulator effects, including changes in survival-promoting MCL1L transcripts, splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket, and self-renewal-promoting ADAR1 and STAT3beta transcripts.

[0118] Here, we describe targeted CSC eradication and unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR, a novel lentiviral splicing reporter assay, and RNA sequencing analysis and used as putative biomarkers to monitor molecular response to 17S-FD-895 treatment. A subset of these transcripts can only be detected after exposure to splicing modulators, and their generation occurs prior to cytotoxic effects in cells, thus allowing quantification of relative sensitivity and estimation of response to splicing modulation. Together, these molecular tools provide a sensitive method to detect the activity and mechanism of action of 17S-FD-895 and clearly demonstrate the CSC selectivity of 17S-FD-895 in humanized stromal co-cultures and humanized CSC mouse models, which has utility in the future clinical development of this class of therapeutic agent.

[0119] In addition to the on-target splicing modulator effects detected in human and rat leukemia cells treated with 17S-FD-895, we also found that human multiple myeloma cells were highly sensitive to spliceosome perturbation. SF3B1 and SF3B3 splice biomarkers, together with MCL-1 transcripts, were the most highly expressed and responsive to 17S-FD-895 in human cells.

[0120] In vitro dose-response splicing modulator treatment and human-specific splice biomarker analysis in primary sAML patient samples, we then tested the molecular response on CD34+ CSCs isolated from primary sAML patient samples treated with 17S-FD-895 in vitro.

[0121] We used lentiviral splicing reporter in human AML CSCs for in vitro splicing modulator treatment and detection of intron retention in living cells. To detect splicing activity in live primary human AML CSCs, we first transduced human AML CSCs (CD34+ cells) with lentiviral fluorescent splicing reporter. Consistent with the results observed in stably transduced human leukemia cell lines, human AML CSC cells treated with 17S-FD-895 showed increased intron retention after 17S-FD-895 treatment.

[0122] We next determined the effect of splicing modulation in vivo on sAML CSC burden in a clinically relevant animal model, 2018-PDX50261-IV-2 (a non-mutated sAML PDX model). We tested increased dosing frequency at a previously used concentration (10 mg / kg) of 17S-FD-895 to evaluate CSC burden after treatment in sAML-engrafted mice. Furthermore, we determined the effect of splicing modulation on sAML CSC burden in a clinically relevant animal model, 2019-PDX2008-5-I. High-sensitivity splice biomarker analysis using primers specific for human transcripts in qRT-PCR assays clearly demonstrated that in vivo 17S-FD-895 treatment induced intron retention of SF3B family and MCL1 transcripts and reduced expression of sAML CSC-specific transcripts. These biomarker studies provide evidence of on-target molecular responses to splicing modulator treatment that can be rapidly quantified and monitored in human cells isolated from leukemia-engrafted animal tissues. This suggests the potential utility of these molecular assays as paired biomarkers in a clinical setting. For human-specific biomarker detection and in vivo dose modeling, we completed splice biomarker studies on cells isolated from sAML PDX50261 mice treated with 17S-FD-895 at doses of 10-20 mg / kg compared to vehicle control. Splice biomarker analysis in dose-escalation studies showed a pattern of exon skipping (MCL1-S expression) consistent with on-target splicing modulator activity of 17S-FD-895, along with increased MCL1-S / L ratios and SF3B family member intron retention (most notably in the SF3B3 transcript). No evidence of test article-related toxicity was observed after 2 weeks of dosing at 20 mg / kg in this humanized in vivo model of sAML. Overall, a dose of 10 mg / kg in mice is sufficient to reduce sAML CSC burden in vivo while sparing normal HSPC development.No evidence of test article-related toxicity was observed after 2 weeks of dosing at 10 or 20 mg / kg in a humanized in vivo model of normal HSPC development. Our biomarker system confirmed the functional and molecular therapeutic index of 17S-FD-895, with sAML CSCs being more sensitive to splicing regulation than normal HSPCs and their progeny.

[0123] To further investigate the role of pre-mRNA splicing in pediatric AML stem and progenitor cells, we performed RNAseq of highly purified non-leukemic or AML hematopoietic stem cells (HSCs; CD34+CD38-Lin-) and hematopoietic progenitor cells (HPCs; CD34+CD38+Lin-) from pediatric bone marrow or peripheral blood. Utilizing a splice variant-specific alignment algorithm, we assessed genome-wide alternative splicing events in HSCs and HPCs derived from pediatric patients with AML. We observed differential splicing of all classes of alternative splicing events, including cassette exon skipping, intron retention, and 5' and 3' splice site competition. MCL1-L, a pro-survival splice variant of the BCL2 family, was shown to be highly expressed in pediatric AML progenitors via RNA sequencing and was significantly increased between CD34+ cord blood and pediatric AML samples via qRT-PCR.HSCs show decreased expression levels of RBFOX2 and both MBNL1 and MBNL2, whereas AML-derived HPCs suggest antagonistic co-regulation of splicing by RBFOX2 and CELF2.

[0124] We used qPCR to evaluate the effect of this splicing modulator on pro-survival splice variants in CD34+ cells obtained from both peripheral blood and bone marrow of pediatric AML patients. PCR revealed a dose-dependent increase in MCL1 exon 2 skipping, generating pro-apoptotic MCL1-S transcripts. Furthermore, splicing modulation induces an increase in SF3B1 intron levels and a decrease in misspliced ​​PTK2B-202 after treatment. Furthermore, hematopoietic progenitor cell assays revealed a dose-dependent decrease in clonogenicity and self-renewal in CD34+ cells isolated from pediatric AML samples. Notably, pediatric AML samples were more sensitive to splicing modulation than both de novo AML and sAML, whereas normal CB samples were not affected by splicing modulator treatment.

[0125] We performed further pharmacokinetic, safety and tolerability studies in rats, rabbits and monkeys. In animal toxicology studies, 17S-FD-895 was well tolerated at the doses tested. The toxicology studies included non-GLP single-dose tolerability and TK studies in rats, rabbits and monkeys. In rats, dose-dependent changes in blood parameters were observed, including decreases in mean neutrophil, monocyte, eosinophil and platelet counts. In rabbits, there was a moderate decrease in several blood parameters, including platelets. In monkeys, there was a minimal decrease in platelet counts in a dose-dependent manner. There were no other significant toxicological findings in the in vivo animal studies.

[0126] Previous toxicology studies included non-GLP single-dose tolerability and TK in rats, rabbits, and monkeys. In rats, minimal and statistically non-significant reductions in monocytes, neutrophils, and eosinophils were observed after administration of 17S-FD-895. At doses ≥20 mg / kg 17S-FD-895, there was a minimal reduction in platelets in female rabbits. In monkeys, no changes in hematology, coagulation, or clinical chemistry parameters were observed. There were no other significant toxicology findings in in vivo animal studies. Future studies include non-GLP dose range-finding studies in rats and rabbits, as well as GLP-definite tolerability / TK studies in rats and rabbits with a clinically relevant route (IV) and schedule (twice weekly for 4 doses). Previous IND-enabling pharmacology studies provided in vivo CSC selectivity data, clearly showing that 17S-FD-895 treatment reduces the frequency of CSCs in treatment-resistant secondary AML. We identified sensitive splice biomarkers of in vivo response to splicing modulation with 17S-FD-895 and showed that treatment with 17S-FD-895 resulted in a significant increase in intron retention (SF3B1), exon skipping (MCL1-S), along with a decrease in sAML CSC-specific functional splice biomarker (CD44-012).Previous studies have also shown that 17S-FD-895 specifically targets CSCs rather than bulk populations in sAML, sparing normal hematopoietic stem and progenitor cells, clearly demonstrating that the effect of 17S-FD-895 is based on spliceosome deregulation, independent of splicing factor mutation status.

[0127] Finally, PD studies in PBMCs from monkeys receiving 17S-FD-895 demonstrated on-target splicing modulation exemplified by MCL1 exon skipping and SF3B3 intron retention.

[0128] In one study, the patient was a 72 year old female diagnosed with AML, untreated, with: Mutations: CEBPA, CSF3R, DNMT3A, DNMT3B, EP300, ETV6, EZH2, FANCL, KMT2C, LUC7L2, NOTCH1, NRAS, RPL5, SUZ12, TET2. Cytogeneics: 46,XX,1,inv(3)(q21q26.2),del(5)(q14q34),der(12 )t(1;1 2)(q21;p11.2),20,+r,+mar1[9] / 46,sl,der(7)t(7;9)(p13;q13)[4] / 46,sl,i(21)(q10)[3 ] / 46,sl, add(2)(q31)[2] / 46,sl,add(2)(q33)[2]

[0129] Also in this study, the patient was a 68 year old male diagnosed with MF and high risk for AIHA, treated with ruxolitinib, and had the following: Mutations: MPL (47%), TET2 (42%), KMT2C (49%), SRSF2 (50%), VCUS: MPL R.V501M (49%), PAX5 (50%). Cytogenetics: unbalanced translocation (1;6) with gain of 1q, loss of 6p; loss of 20q (in 4% of cells). In this study, revexinib (17S-FD-895) significantly inhibited CD34+Lin- cells in the sAML50261-implanted NSG-SGM3 mouse model, as shown graphically in Figure 1A-C.

[0130] References Example 1: [ka] [ka]

[0131] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. (a) Rebeccinibe, also known as 17S-FD-895 【Chemistry 5】 or an enantiomer, stereoisomer, deuterated version, or salt thereof; and (b) at least one second drug; 20. A pharmaceutical or therapeutic composition, formulation or therapeutic combination of drugs comprising:

2. - the treatment and amelioration of cancer, optionally wherein said cancer is leukemia, optionally wherein said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; - inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSC) in vivo; - in vivo inhibition of the spliceosome in AML or MPN, and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); 2. A formulation, pharmaceutical composition or therapeutic combination of drugs for the treatment of a disease comprising: (a) Rebeccinibe, also known as 17S-FD-895; 【Chemistry 6】 or an enantiomer, stereoisomer, deuterated version, or salt thereof, or (b) the compound of (a), or revexinib or 17S-FD-895, and at least one second drug; 23. A formulation, pharmaceutical composition or therapeutic combination of drugs comprising:

3. 3. The pharmaceutical or therapeutic composition, formulation or therapeutic combination of claim 1 or 2, characterized in that the dose of 17S-FD-895 is administered or formulated for administration once daily for 1 to 2 weeks, twice weekly for 2 weeks or about 1 to 2 weeks, followed by 2 weeks off or 2 to 4 weeks off, continuing for 2, 3, 4, 5 or 6 cycles, optionally continuing for four 28 day cycles or monthly cycles.

4. The at least one second drug comprises an ATP-competitive protein tyrosine kinase inhibitor, optionally wherein the ATP-competitive protein tyrosine kinase inhibitor is selected from the group consisting of dasatinib (or SPRYCEL ＴＭ Or DASANIX ＴＭ 4. The pharmaceutical or therapeutic composition, formulation or drug therapeutic combination of any one of claims 1 to 3, comprising

5. The at least one second drug is a JAK2 (Janus kinase 2) inhibitor, optionally fedratinib (or INREBIC ＴＭ 4. The pharmaceutical or therapeutic composition, formulation or therapeutic combination of any one of claims 1 to 3, comprising:

6. The at least one second drug comprises a chemotherapeutic agent, and optionally the chemotherapeutic agent is afatinib (or GILOTRIF ＴＭ ), afuresertib, alectinib, alisertib, alvocidib, amsacrine, amonafide, amuvatinib, axitinib, azacitidine, azathioprine, bafetinib, barasertib, bendamustine, bleomycin, bosutinib, bortezomib, busulfan, cabozantinib, camptothecin, canertinib, capecitabine, cabazitaxel, carboplatin, carmustine, cenisertib, ceritinib, chlorambucil, cisplatin, cladoxaminib, cyclosporine ... Livin, clofarabine, crenolanib, crizotinib, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dacomitinib, dactinomycin, danusertib, dasatinib, daunorubicin, decitabine, dinaciclib, docetaxel, dovitinib, doxorubicin, epirubicin, epitinib, eribulin mesylate, erlotinib, etirinotecan, etoposide, everolimus, exemestane, fedratinib (or INREBIC ＴＭ ), floxuridine, fludarabine, fluorouracil, gefitinib, gemcitabine, hydroxyurea, ibrutinib, icotinib, idarubicin, ifosfamide, imatinib, ipatasertib, irinotecan, ixabepilone, lapatinib, lenalidomide, lestaurtinib, lomustine, lucitanib, masitinib, mechlorethamine, melphalan, mercaptopurine, methotrexate, midostaurin, mitomycin, mitoxantrone , mubritinib, nelarabine, neratinib, nilotinib, nintedanib, omacetaxine mepesuxinate, orantinib, oxaliplatin, paclitaxel, palbociclib, palifosfamide tris, pazopanib, pelitinib, pemetrexed, pentostatin, plicamycin, ponatinib, poziotinib, pralatrexate, procarbazine, quizartinib, raltitrexed, regorafenib, ruxolitinib (or OPZELURA ＴＭ ), seliciclib, sorafenib (or NEXAVAR ＴＭ ), streptozocin, surufatinib, sunitinib (or SUTENT ＴＭ ), Tamoxifen (or NOLVADEX ＴＭ ), tandutinib, temozolomide, temsirolimus, teniposide, triatinib, thioguanine, thiotepa, topotecan, uramustine, valrubicin, vandetanib, vemurafenib (or ZELBORAE) ＴＭ ), vincristine (or ONCOVIN ＴＭ ), vinblastine (or VELBAN ＴＭ ), vinorelbine (or NAVELBINE ＴＭ ), and vindesine (or ercidin).

7. The at least one second drug comprises a hypomethylating agent (HMA), optionally wherein the HMA is azacitidine (or VIDAZA). ＴＭ ) or decitabine (or DACOGEN ＴＭ 13. A pharmaceutical or therapeutic composition, formulation or therapeutic combination of a drug according to any of the preceding claims comprising:

8. The at least one second drug comprises a second telomerase inhibitor, optionally wherein the telomerase inhibitor is selected from the group consisting of imetelstat, zidovudine (or azidothymidine (AZT)), stavudine (or ZERIT), ＴＭ ), tenofovir or tenofovir disoproxil (or VIREAD ＴＭ ), didanosine (or VIDEX ＴＭ ), abacavir (ZIAGEN ＴＭ 5. The pharmaceutical or therapeutic composition, formulation or therapeutic combination of any of the preceding claims comprising at least one, two or three of: TMPI, Telomestatin, RHPS4, BRACO-19, TMPyP4, Tertomotide, ASTVAC-1, GX-301, UCPVax, UV-1, Vx-001, Vx-006, INO-1400, INVAC-1, ASTVAC-2, Telin (ab 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone), Vbx-011, Vbx-021, Vbx-026, INO-5401, KML-001, TK-005, Ribovax, Vbx-016, ZI-HX, ZI-H04 and ZIH-03.

9. The formulation, pharmaceutical composition or therapeutic drug combination, or the active agent or drug contained therein, is, is formulated in, or is contained in, a liquid formulation (optionally sterile saline or water), a spray, powder, aerosol, mist, or any formulation for inhalation, pill, capsule, tablet, or gel tab, or equivalent); or is coated on, or is contained in, a bead, powder, particle, or multi-layered bead or particle, optionally the bead, powder, particle, or multi-layered bead or particle is formulated in, or is contained in, a pill, capsule, tablet, or gel tab, or equivalent, for oral delivery.

9. The pharmaceutical or therapeutic composition, formulation or therapeutic combination of drugs of claims 1-8, wherein optionally for oral delivery said pill, capsule, tablet, gel tab or equivalent is a hard gelatin capsule or equivalent or comprises hard gelatin or equivalent; or a drug delivery device or package, blister pack, clamshell or tray comprises a plurality of compartments spatially arranged on said drug delivery device or package, blister pack, clamshell or tray to follow a dosage regimen.

10. 10. The pharmaceutical or therapeutic composition, formulation or therapeutic combination of any of claims 1 to 9, characterized in that the active agent or drug in said formulation, pharmaceutical composition or therapeutic combination of drug is dosed at between about 10-500 mg / day or between about 500-1 g per day, or at a dosage of between about 100-600 mg per day or per dosage, or at about 100 mg, 200 mg, 300 mg, 400 mg, 500 mg or 600 mg per day or per dosage, and optionally the unit dosage is administered once per day (QD), or twice per day (BID), or three times per day (TID), or more, to an individual in need of administration.

11. 11. A pharmaceutical or therapeutic composition, formulation or therapeutic combination of a drug according to any of claims 1 to 10, characterized in that the active agent or drug in said formulation, pharmaceutical composition or therapeutic combination of a drug is administered as or formulated with or as an inhaled or aerosol formulation (e.g. a powder or mist or aerosol) and / or is formulated with or as an oral, intramuscular (IM), subcutaneous (SC), intrathecal or intravenous (IV) formulation, whereby optionally said inhaled (or aerosol) formulation and said oral, IV, SC, intrathecal and / or IM formulation are both administered simultaneously or sequentially.

12. The formulation, pharmaceutical composition or therapeutic combination of drugs comprises: a drug delivery device is optionally used by inhalation, wherein said drug delivery device optionally comprises an inhalation device or inhaler or a nasal spray device, optionally said inhaler or nasal spray device is a hand-held inhaler or nasal spray device, optionally said inhaler or nasal spray device is a metered or metered dose inhaler or nasal spray device, or Intravenously (IV) or intramuscularly (IM), Administered to an individual in need thereof, A pharmaceutical or therapeutic composition, formulation or drug therapeutic combination according to any one of claims 1 to 11.

13. - the treatment and amelioration of cancer, optionally wherein the cancer is leukemia, optionally wherein the cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; or - inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSC) in vivo; - in vivo inhibition of the spliceosome in AML or MPN, and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); A pharmaceutical composition for use in A pharmaceutical composition comprising 17S-FD-895 (or revexinib), or 17S-FD-895 and at least one second drug.

14. - the treatment and amelioration of cancer, optionally wherein said cancer is leukemia, optionally wherein said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; or - inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSC) in vivo; - in vivo inhibition of the spliceosome in AML or MPN, and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); 23. A composition for use in the manufacture of a medicament or pharmaceutical composition for the treatment of chronic obstructive pulmonary disease, comprising 17S-FD-895 (or revexinib), or 17S-FD-895 and at least one second drug.

15. - the treatment and amelioration of cancer, optionally wherein said cancer is leukemia, optionally wherein said cancer is acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and / or multiple myeloma (MM); - inhibition of myeloproliferative neoplasm (MPN) or AML stem cell proliferation in vivo; or - inhibition of pre-leukemic stem cell (pre-LSC) transformation into leukemic stem cells (LSC) in vivo; - in vivo inhibition of the spliceosome in AML or MPN, and / or - Inhibition in vivo of transcripts that bind to components of the spliceosome binding pocket or that bind to ADAR1 (adenosine deaminase acting on RNA-1); 23. Use of 17S-FD-895 (or revexinib), or 17S-FD-895 and at least one second drug, in the manufacture of a medicament or pharmaceutical composition for

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