Methods and compositions for the treatment of blood cancer

Abstract

Described herein are methods relating to the treatment of a blood cancer by administration of a RhoA / ROCK inhibitor, ROCK1 inhibitor, and / or JAK2 inhibitor.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF BLOOD CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 604,227 filed November 30, 2023, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The technology described herein relates to the treatment of blood cancer, e.g., myeloprolifertive neoplasms.GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant No. HL161175 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Myeloproliferative neoplasms (MPNs) are a group of blood cancers, in which bone marrow cells develop and function abnormally. In patients with primary and secondary myelofibrosis, immature megakaryocytes (MKs) accumulate in the bone marrow. These immature MKs release cytokines into the bone marrow that cause fibrosis. This fibrosis leads to leads to low levels of mature blood cells, which is the major cause of mortality in patients with myelofibrosis.

[0005] Currently, it is not understood how these immature MKs secrete the cytokines that cause fibrosis. While current treatments aim at preventing megakaryocytosis and MK differentiation defects in myelofibrosis, there is a need to find other treatment options, and the ability to target cytokine release from MKs would improve treatment and patient outcomes.SUMMARY

[0006] Immature megakaryocytes (MKs) release pro-fibrotic cytokines into the bone marrow, causing fibrotic alterations that lead to blood cancers, such as myeloproliferative neoplasms (MPN). The inventors have now found that treatment with RhoA / ROCK inhibitors has the surprising effect of normalizing cytokine release and preventing fibrotic alterations in myelofibrosis. These RhoA / ROCK inhibitors directly inhibit autophagy and cytokine release by the immature MKs. Accordingly, described herein are methods of treating a blood cancer, e.g. a myeloproliferative neoplasm, relating to this surprising effect of preventing or reducing pro-fibrotic cytokine release using RhoA / ROCK inhibitors.

[0007] In one aspect of any of the embodiments, described herein is a method of treating blood cell cancer in a subject in need thereof, the method comprising administering to the subject a RhoA / ROCK inhibitor.

[0008] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; RKI-1447; Y-27632, GSK429286A; Y-30141; AT-13148; BA-210; [3-elemene; belumosudil; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; ibuprofen; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y- 30141; Y-33075; and Y 33983. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is belumosudil, netarsudil, ripasudil, or fasudil. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is belumosudil or netarsudil.

[0009] In some embodiments of any of the aspects, the subject has or is diagnosed as having a myeloproliferative neoplasm. In some embodiments of any of the aspects, the subject has or is diagnosed as having primary myelofibrosis or secondary myelofibrosis. In some embodiments of any of the aspects,

[0010] In some embodiments of any of the aspects, the method further comprises administering to the subject a JAK2 inhibitor. In some embodiments of any of the aspects, the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; filgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868. In some embodiments of any of the aspects, the JAK2 inhibitor is ruxolitinib.

[0011] In some embodiments of any of the aspects, the subject is a mammal. In some embodiments of any of the aspects, the subject is a human.

[0012] In some embodiments of any of the aspects, the amount and / or rate of myelofibrosis in the subject is reduced. In some embodiments of any of the aspects, the amount and / or rate of myelofibrosis in the bone marrow and / or spleen of the subject is reduced.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 depicts a schematic of MK secretion in health and disease. Left: Release of TGF[31 and CXCL4 maintains HSC quiescence in healthy marrow.34721Right: Immature, aberrant MKs in myelofibrosis affect BM homeostasis by enhanced secretion of soluble mediators, which influence osteoclastogenesis,22,23enhance osteoblast proli-feration24,25and ultimately induce collagen I and reticulin deposition.26Image was created using biorender.com. References are provided in Example 3.

[0014] Fig. 2. Regulation and targeting of autophagy in MKs. Besides conventional release of proteins, cargo can be processed within the autophagic machinery. Nutrient starvation induces activity of mTOR, which is enhanced by rapamycin. Degradative auto-phagy depends on initial autophagosome formation, followed by their fusion with lysosomes. Release of autophagosomes from the plasma membrane relies on Atg proteins such as LC3B, and involves cytoskeletal regulators such as RhoA (inhibited by Rhosin, fasudil) and the kinase ULK1 and can be influenced by the small GTPase Arf6, which is also important for dynamin 2-mediated endocytosis and affects activity ofPI3K (inhibited by 3 -methyladenine). Image was created using biorender.com. ER: endoplasmic reticulum; PI3K: phosphoinositide-3-kinase, ROCK: Rho kinase.

[0015] Figs. 3A-3F demonstrate that mutation within Mpig6b leads to macrothrombocytopenia, myelofibrosis and sex-specific osteosclerosis. Platelet count (Fig. 3A) and size (Fig. 3B) in wildtype (WT) and Mpig6bmut mice were assessed at an automated blood cell analyzer. (Fig. 3C) BM paraffin sections ofWT and Mpig6bmut mice reveal sex-specific osteosclerosis. Scale bars: 100 pm. (Fig. 3D) Cryosections of femoral bones from WT and Mpig6bmut mice were stained for collagen IV (Col4), CD105 and GPIX and analyzed by confocal microscopy. Scale bars: 500 pm. (Fig. 3E) BM MKs and vessels in whole femora cryosections were counted using ImageJ. (Fig. 3F) Levels of TGFJ31 in BM plasma of female and male WT and Mpig6bmut mice were determined using enzyme-linked immunosorbent assays. Values are mean ± SD. *P <0.05; **P < 0.01; ***P < 0.001.

[0016] Figs. 4A-4B demonstrate the role of RhoA in myelofibrosis. (Fig. 4A) Reticulin fibers in spleen and BM paraffin sections of WT, Mpig6bmut and RhoA- / - / Mpig6bmut mice Scale bars: 75 pm. (Fig. 4B) Levels of TGFJ31 in BM plasma of RhoA+ / + , Mpig6bmut and RhoA- / - / Mpig6bmut mice were determined using enzyme-linked immunosorbent assays. Values are mean ± SD. *P < 0.05;**P < 0.01.

[0017] Fig. 5 depicts femoral cryosections of WT and conditional Arf6- / - mice stained for collagen I, GPIX and CD105 were imaged by confocal microscopy (Zeiss LSM880; lOx objective). Scale bars: 500 pm.

[0018] Fig. 6 depicts MK within the BM microenvironment. Transmission electron micrograph of a BM MK interacting with a variety of cell types within the BM. The MK cytoplasm is encircled in black. White arrows point to BM cells in close vicinity to the mature MK, which is also localized.DETAILED DESCRIPTION

[0019] The inventors have surprisingly found that RhoA / ROCK inhibitors can normalize cytokine release and reduce fibrosis in blood cancer, e.g., myelofibrosis. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating a blood cancer or blood cell cancer in a subject in need thereof, the method comprising administering to the subject at least one RhoA / ROCK inhibitor. In one aspect of any of the embodiments, described herein is at least RhoA / ROCK inhibitor for use in a method of treating a blood cancer or blood cell cancer and / or for use in manufacture of a medicament for treating a blood cancer.

[0020] As used herein “Ras homolog gene family, member A (RhoA) / Rho-associated protein kinase (ROCK)” refers to a pathway in which RhoA activates one or both ROCKs. RhoA is a small GTPase protein in the Rho family comprising an effector domain, four exons, a hypervariable region and a CAAX box motif. Sequences for RhoA in a number of species are known, e.g., human RhoA (NCBI Gene ID: 387). The small GTP-binding proteins of the Rho family are involved in theregulation of various aspects of cell motility, shape, proliferation and apoptosis. Rho kinases, also referred to as ROCKs, are serine / threonine kinases activated by GTP-bound Rho proteins that phosphorylate downstream targets in the ROCK pathway. Sequences for ROCKs in a number of species are known, e.g., human R0CK1 (NCBI Gene ID: 6093) and ROCK2 (NCBI Gene ID: 9475). Phosphorylation targets include, but are not limited to myosin light chain phosphatase, LIM kinases, adducin, and ezrin-radixon-moesin (ERM) proteins. Other or related Rho kinase functions include, for example, regulation of smooth muscle cell contraction, cell migration, and maintenance of cell viability and morphology, in part by regulating stress fibers and focal adhesions.

[0021] As used herein, “inhibitor” refers to an agent which can decrease the expression and / or activity of a target, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more. The efficacy of an inhibitor of one or more targets, e.g. its ability to decrease the level and / or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and / or the activity of the target. In some embodiments of any of the aspects, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule. An inhibitor of a target described herein can inhibit the activity, expression, or accumulation of the target polypeptide. Inhibitors can include inhibitors that act directly on the target itself (e.g., that bind to the protein or transcript, e.g., direct inhibitors). In some embodiments of any of the aspects, an inhibitor of a specified target is an antibody, antibody reagent, or antigen-binding fragment thereof, that specifically binds to the target.

[0022] As used herein, the terms “RhoA / ROCK inhibitor,” “ROCK inhibitor,” and “RhoA inhibitor” are used interchangeably and refer to an inhibitor of one or more Rho-associated Kinases (ROCKs). ROCKs are serine-threonine kinases that act on the cytoskeleton. ROCKs include ROCK1 and ROCK2 and sequences for both are known for a variety of species, e.g., human ROCK1 (NCBI Gene ID No: 6093) and human ROCK2 (NCBI Gene ID No: 9475). ROCK activity can be measured using methods well known in the art, e.g., commercially available kits such as Cat. No. STA-416 from Cell BioLabs (San Diego, CA). ROCK activity and inhibitors are further discussed, e.g., in Barcelo et al. Trends in Cancer 9(3):P250-263 (2023): Liao et al. J Caridovasc Pharmacol 50: 17-24 (2007); Peng et al. Journal of Medicinal Chemistry 59:2269-2300 (2016); each of which is incorporated by reference herein in its entirety.

[0023] ROCK inhibitors can be specific for either ROCK1 or ROCK2 or inhibit both ROCK1 and ROCK2. Non-limiting examples of ROCK inhibitors can include fasudil; ripasudil; netarsudil; RKI-1447; Y -27632, GSK429286A; Y-30141; AT-13148; BA-210; [3-elemene; belumosudil; chroman 1; DJ4; GSK-576371; H-1152; hydroxy fasudil; ibuprofen; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; Y 33983; Wf-536; an azabenzimidazole- aminofurazan, DE- 104, an olefin; an isoquinoline; an indazole; a pyridinealkene derivative; H-l 152; an ROKa inhibitor (BF); XD-4000; HMN-1152; a 4-(l-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamide; rhostatin; BA-207; BA-215; BA-285; BA-1037; Ki-23095; VAS-012; and a quinazoline. In some embodiments, the RhoA / ROCK inhibitor is fasudil; ripasudil; netarsudil; RKI- 1447; Y -27632, GSK429286A; Y-30141; AT-13148; BA-210; p-elemene; belumosudil; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; ibuprofen; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and / or Y 33983. In some embodiments, the RhoA / ROCK inhibitor is belumosudil, netarsudil, ripasudil, or fasudil. In some embodiments, the RhoA / ROCK inhibitor is belumosudil or netarsudil.

[0024] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; belumosudil; RKI-1447; Y-27632;GSK429286A; Y-30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; ibuprofen; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y- 30141; Y-33075; and Y 33983.

[0025] In some embodiments, the RhoA / ROCK inhibitor is fasudil; ripasudil; netarsudil; RKI- 1447; Y-27632, GSK429286A; Y-30141; AT-13148; BA-210; p-elemene; belumosudil; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and / or Y 33983. In some embodiments, the RhoA / ROCK inhibitor is belumosudil, netarsudil, ripasudil, or fasudil. In some embodiments, the RhoA / ROCK inhibitor is belumosudil or netarsudil. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; belumosudil; RKI- 1447; Y-27632; GSK429286A; Y-30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK- 576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.

[0026] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: RKI-1447; Y-27632; GSK429286A; Y-30141; AT-13148; BA-210; p- elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; ibuprofen; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: RKI-1447; Y-27632; GSK429286A; Y-30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.

[0027] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: ripasudil; netarsudil; RKI-1447; Y-27632; GSK429286A; Y-30141; AT- 13148; BA-210; P-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; ibuprofen; LX- 7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: ripasudil; netarsudil; RKI-1447; Y-27632; GSK429286A; Y-30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI- 1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.

[0028] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: Netarsudil; Y-30141; BA-210; P-elemene; GSK-576371; H-1152; Ibuprofen; Y-33075; Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001; GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677; LX-7101; GSK180736; Y-27632; Y- 27632 dihydrochloride; Fasudil; TS-f22; Hydroxyfasudil; Belumosudil; and NRL-1049. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: Netarsudil; Y-30141; BA-210; p-elemene; GSK-576371; H-1152; Y-33075; Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001; GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622; GSK429286A;RKI-1447; Ripasudil; SR3677; LX-7101; GSK180736; Y-27632; Y-27632 dihydrochloride; Fasudil; TS-f22; Hydroxyfasudil; Belumosudil; and NRL-1049.

[0029] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: Netarsudil; Y-30141; BA-210; P-elemene; GSK-576371; H-1152; Ibuprofen; Y-33075; Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001; GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677; LX-7101; GSK180736; Y-27632; Y- 27632 dihydrochloride; TS-f22; Hydroxyfasudil; and NRL-1049. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is selected from the group consisting of: Netarsudil; Y-30141; BA-210; P-elemene; GSK-576371; H-1152; Y-33075; Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001; GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677; LX- 7101; GSK180736; Y-27632; Y-27632 dihydrochloride; TS-f22; Hydroxyfasudil; and NRL-1049.

[0030] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is belumosudil, netarsudil, ripasudil, or fasudil. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is belumosudil or netarsudil. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is netarsudil. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is ripasudil. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is netarsudil or ripasudil.

[0031] The structures and methods of making such RhoA / ROCK inhibitors are known in the art and many are commercially available. For further discussion of such inhibitors, see, e.g., e.g., in Liao et al. J Caridovasc Pharmacol 50: 17-24 (2007); Feng et al. Journal of Medicinal Chemistry 59:2269- 2300 (2016); each of which is incorporated by reference herein in its entirety.

[0032] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is a ROCK1 inhibitor, i.e., it inhibits at least ROCK1. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is a ROCK1 / 2 inhibitor, i.e., it inhibits at least ROCK1 and ROCK2.

[0033] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is a ROCK1 specific inhibitor, i.e., it inhibits only ROCK1. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is a ROCK1 / 2 specific inhibitor, i.e., it inhibits only ROCK1 and ROCK2.

[0034] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is a ROCK1 preferential inhibitor, i.e., it inhibits ROCK1 more strongly than any other target. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor is a ROCK1 / 2 preferential inhibitor, i.e., it inhibits ROCK1 and ROCK2 more strongly than any other target.

[0035] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is selected from the group consisting of: chroman 1; BAY-549; TCS- 7001; GSK269962A; verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622GSK429286A; RKI-1447; ripasudil; SR3677; GSK180736; and Y-27632.

[0036] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is Y-27632. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor, is Y-27632. In some embodiments of any of the aspects, the ROCK1 inhibitor is Y-27632. In some embodiments of any of the aspects, the ROCK1 / 2 inhibitor is Y-27632.

[0037] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 300 nM or lower. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 250 nM or lower. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 220 nM or lower. The lower the IC50, the greater the inhibitory effect.

[0038] In some embodiments of any of the aspects, the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 50 pM to 300 nM. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 50 pM to 250 nM. In some embodiments of any of the aspects, the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 50 pM to 220 nM.

[0039] Table 1

[0040] In some embodiments of any of the aspects, the method further comprises administering to the subject at least one JAK2 inhibitor. In one aspect of any of the embodiments, described herein is at least one RhoA / ROCK inhibitor and at least one JAK2 inhibitor for use in a method of treating a blood cancer or blood cell cancer and / or for use in manufacture of a medicament for treating a blood cancer.

[0041] In some embodiments of any of the aspects, the method further comprises administering to a subject with blood cancer, e.g., MPN, a Janus kinase (JAK) inhibitor. In some embodiments of any of the aspects, the JAK inhibitor is a JAK2 inhibitor.

[0042] As used herein, a Janus Kinase (JAK) inhibitor is a type of immune modulating medication which inhibits the activity of one or more of the Janus kinase family of enzymes (JAK1, JAK2, JAK3, and TYK2). Janus Kinase (JAK) proteins are comprised of four main domains. There is a FERM domain, which exists at the N-terminus and is important for JAKs binding to cytokine receptors, a Src2 -homology-2 (SH2) domain, a pseudo-kinase domain, and a kinase domain at the C- terminus. This domain is responsible for the enzymatic activity of the kinase.

[0043] JAK inhibitors (e.g., JAK2 inhibitors) can be divided into non-selective and selective inhibitors of JAKs (e.g., JAK2). They can also be classified based on their binding mode and type of interactions with the amino acids in JAKs (e.g., JAK2) into reversible (competitive) and irreversible (covalent) inhibitors.

[0044] Competitive JAK inhibitors (e.g., JAK2 inhibitors) form reversible (non-covalent) binding interactions with the amino acids in the four JAKs. The binding interactions formed by this type of JAK inhibitors (e.g., JAK2 inhibitors) include hydrogen bonds and hydrophobic interactions. The class of reversible JAK inhibitors (e.g., JAK2 inhibitors) can also be classified into two subclasses: Type I JAK inhibitors (e.g., JAK2 inhibitors) and Type II JAK inhibitors (e.g., JAK2 inhibitors).

[0045] Type I JAK inhibitors (e.g., JAK2 inhibitors) bind to the ATP-binding site of the JAKs (e.g., JAK2 inhibitors) under the active conformation of the kinase domain. This includes clinically approved drugs such as filgotinib, which acts and is classified as a selective JAK1 inhibitor, while fedratinib exhibits selective inhibition of JAK2. On the other hand, tofacitinib and peficitinib act by blocking multiple JAKs. Without wishing to be bound by theory, the ability of type I JAK inhibitors (e.g., JAK2 inhibitors) to bind to multiple kinases and act as non-selective inhibitors could be due to the highly conserved structure of the ATP-binding site in the four JAKs.

[0046] Type II JAK inhibitors (e.g., JAK2 inhibitors) also bind to the ATP-binding site of the kinase domain in the inactive conformation of JAKs. NVP-BBT594 and NVP-CHZ868 are representative examples of type II inhibitor, which target JAK2.

[0047] The allosteric JAK inhibitors (e.g., JAK2 inhibitors) include small molecule inhibitors that bind to a site other than the ATP-binding site in JAKs. Among these inhibitors, deucravacitinib (BMS-986165) act as a selective allosteric inhibitor of TYK2. In addition, LS104, and ON044580 are examples of JAK2 allosteric inhibitors. Additional information regarding the groups of JAK inhibitors can be found in Shawky AM, Almalki FA, Abdalla AN, Abdelazeem AH, Gouda AM. A Comprehensive Overview of Globally Approved JAK Inhibitors. Pharmaceutics. 2022 May6;14(5): 1001. doi: 10.3390 / pharmaceuticsl4051001. PMID: 35631587; PMCID: PMC9146299, which is incorporated by reference herein in its entirety.

[0048] JAK inhibitors (e.g., JAK2 inhibitors) that target the JAK proteins include, but are not limited to, ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; fdgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868. In some embodiments of any of the aspects, the JAK inhibitor is a JAK2 inhibitor. In some embodiments of any of the aspects, the JAK inhibitor inhibits at least JAK2. In some embodiments of any of the aspects, the JAK inhibitor is a JAK2- specific inhibitor, i.e., it inhibits only JAK2 and not other JAKs. In some embodiments of any of the aspects, the JAK inhibitor is a JAK2 -preferential inhibitor, e.g., it inhibits JAK2 more strongly than other JAKs..In some embodiments of any of the aspects, the JAK2 inhibitor is ruxolitinib.

[0049] Additional information on structure of JAK inhibitors (e.g., JAK2 inhibitors) and administration of JAK inhibitors (e.g., JAK2 inhibitors) can be found in U.S. Patent 7,031,023, U.S. Patent 8,808,764, U.S. Patent 10,617,690 and U.S. Patent 10,973,913, which are incorporated by reference herein in their entireties.

[0050] In some embodiments of any of the aspects, the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; fdgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868. In some embodiments of any of the aspects, the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; febratinib; pacritinib; and momelotinib. In some embodiments of any of the aspects, the JAK2 inhibitor is ruxolitinib.

[0051] In some embodiments of any of the aspects, one RhoA / ROCK inhibitor is administered to the subject in need thereof. In some embodiments, two RhoA / ROCK inhibitors are administered to the subject in need thereof. In some embodiments, three RhoA / ROCK inhibitors are administered to the subject in need thereof. In some embodiments, four RhoA / ROCK inhibitors are administered to the subject in need thereof. In some embodiments, five RhoA / ROCK inhibitors are administered to the subject in need thereof. In some embodiments, two or more RhoA / ROCK inhibitors are administered to the subject in need thereof. In some embodiments, multiple RhoA / ROCK inhibitors are administered to the subject in need thereof.

[0052] In some embodiments of any of the aspects, one JAK inhibitor is administered to the subject in need thereof. In some embodiments, two JAK inhibitors are administered to the subject in need thereof. In some embodiments, three JAK inhibitors are administered to the subject in need thereof. In some embodiments, four JAK inhibitors are administered to the subject in need thereof. In some embodiments, five JAK inhibitors are administered to the subject in need thereof. In some embodiments, two or more JAK inhibitors are administered to the subject in need thereof. In some embodiments, multiple JAK inhibitors are administered to the subject in need thereof.

[0053] In some embodiments of any of the aspects, one JAK2 inhibitor is administered to the subject in need thereof. In some embodiments, two JAK2 inhibitors are administered to the subject in need thereof. In some embodiments, three JAK2 inhibitors are administered to the subject in need thereof. In some embodiments, four JAK2 inhibitors are administered to the subject in need thereof. In some embodiments, five JAK2 inhibitors are administered to the subject in need thereof. In some embodiments, two or more JAK2 inhibitors are administered to the subject in need thereof. In some embodiments, multiple JAK2 inhibitors are administered to the subject in need thereof.

[0054] In some embodiments of any of the aspects, the method comprises administering to the subject at least one RhoA / ROCK inhibitor and at least one JAK2 inhibitor. In some embodiments of any of the aspects, the method comprises administering to the subject a RhoA / ROCK inhibitor and at least one JAK2 inhibitor. In some embodiments of any of the aspects, the method comprises administering to the subject at least one RhoA / ROCK inhibitor and a JAK2 inhibitor.

[0055] In some embodiments of any of the aspects, the subject in need of treatment for blood cancer, e.g., MPN, is administered at least one RhoA / ROCK inhibitor and at least one JAK inhibitor. In some embodiments, the subject in need of treatment for blood cancer, e.g., MPN, is administered at least one RhoA / ROCK inhibitor before being administered at least one JAK inhibitor. In some embodiments, the subject in need of treatment for blood cancer, e.g., MPN, is administered at least one RhoA / ROCK inhibitor after being administered at least one JAK inhibitor.

[0056] In some embodiments of any of the aspects, the subject in need of treatment for blood cancer, e.g., MPN, is administered at least one RhoA / ROCK inhibitor and at least one JAK2 inhibitor. In some embodiments, the subject in need of treatment for blood cancer, e.g., MPN, is administered at least one RhoA / ROCK inhibitor before being administered at least one JAK2 inhibitor. In some embodiments, the subject in need of treatment for blood cancer, e.g., MPN, is administered at least one RhoA / ROCK inhibitor after being administered at least one JAK2 inhibitor.

[0057] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having blood cancer, e.g., MPN, the method comprises administering at least one RhoA / ROCK inhibitor. In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having blood cancer, e.g., MPN, the method comprises administering at least one RhoA / ROCK inhibitor and at least one JAK inhibitor. In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having blood cancer, e.g., MPN, the method comprises administering at least one RhoA / ROCK inhibitor and at least one JAK2 inhibitor.

[0058] In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor is Y-2763 and the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; febratinib; pacritinib; and momelotinib. In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor is Y-2763 and the JAK2 inhibitor is ruxolitinib. In some embodiments of anyof the aspects, the at least one RhoA / ROCK inhibitor is Y -l' l 63 and the JAK2 inhibitor is febratinib. In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor is Y-2763 and the JAK2 inhibitor is pacritinib. In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor is Y-2763 and the JAK2 inhibitor is momelotinib.

[0059] As used herein, “cancer” refers to a hyperproliferation of cells that have lost normal cellular control, resulting in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Cancers are classified based on the histological type (e.g., the tissue in which they originate) and their primary site (e.g., the location of the body the cancer first develops), and can be a carcinoma, a melanoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. “Cancer” can also refer to a solid tumor. As used herein, the term “tumor” refers to an abnormal growth of cells or tissues, e.g., of malignant type or benign type. “Cancer” can be metastatic, meaning the cancer cells have disseminated from its primary site of origin and migrated to a secondary site.

[0060] In some embodiments, the cancer treated herein is a blood cancer. In some embodiments of any of the aspects, the subject has or is diagnosed as having a blood cancer. Blood cancers are cancers where bone marrow, the site of blood cell production, creates abnormal amounts of red blood cells, white blood cells, or platelets, e.g., excessive or insufficient amounts of blood cells.

[0061] In some embodiments of any of the aspect, the blood cancer is a myeloproliferative neoplasm. In some embodiments of any of the aspects, the subject has or is diagnosed as having a myeloproliferative neoplasm. Myeloproliferative neoplasm (MPNs) are cancers in which cells in the bone marrow develops and functions abnormally. Non-limiting examples of MPNs include Polycythemia vera (PV), Essential thrombocythemia (ET), Myelofibrosis (MF), chronic myelogenous leukemia, chronic neutrophilic leukemia, and chronic eosinophilic leukemia.

[0062] In some embodiments, the cancer treated herein is Myelofibrosis (MF). In some embodiments of any of the aspects, the subject has or is diagnosed as having Myelofibrosis (MF).

[0063] In some embodiments of any of the aspects, the cancer is a blood cell cancer. A blood cell cancer is any cancer of a blood cell.

[0064] In some embodiments of any of the aspects, the subject has or is diagnosed as having aberrant megakaryopoiesis, essential thrombocythemia, or polycythemia vera. In some embodiments of any of the aspects, the subject has or is diagnosed as having aberrant megakaryopoiesis. In some embodiments of any of the aspects, the subject has or is diagnosed as having essential thrombocythemia. In some embodiments of any of the aspects, the subject has or is diagnosed as having polycythemia vera.

[0065] In some embodiments of any of the aspects, the subject has or is diagnosed as having primary myelofibrosis or secondary myelofibrosis. In some embodiments of any of the aspects, the subject has or is diagnosed as having primary myelofibrosis. In some embodiments of any of the aspects, the subject has or is diagnosed as having secondary myelofibrosis.

[0066] Myelofibrosis is a serious bone marrow disorder that disrupts the body's normal production of blood cells. The result is extensive scarring in bone marrow, leading to severe anemia, weakness, fatigue and often an enlarged spleen. Many subjects or patients with myelofibrosis get progressively worse, and some subjects or patients may eventually develop a more serious form of leukemia. Myelofibrosis can occur when blood stem cells (e.g., HSPCs) develop a genetic mutation.

[0067] Primary myelofibrosis, which is sometimes called chronic idiopathic myelofibrosis, occurs in people who have no history of problems with their bone marrow. Several specific gene mutations have been identified in people with myelofibrosis. The most common genes in which myelofibrosis causing mutations occur are the Janus kinase 2 (JAK2) gene, the calreticulin (CALR) gene, and the thrombopoietin receptor (MPL) gene.

[0068] Secondary myelofibrosis is where the condition develops in people who have other bone marrow disorders, such as polycythaemia vera or essential thrombocythaemia.

[0069] Although the cause of myelofibrosis often isn't known, certain factors are known to increase risk. Increased age can be associated with the development of myelofibrosis. Myelofibrosis can affect anyone, but it's most often diagnosed in people older than 50. Patients with another blood cell disorder are at higher risk for developing myelofibrosis. A small portion of people with myelofibrosis develop the condition as a complication of essential thrombocythemia or polycythemia vera. Exposure to certain chemicals can increase the risk for myelofibrosis. Myelofibrosis has been linked to exposure to industrial chemicals such as toluene and benzene. Exposure to radiation can increase the risk for myelofibrosis.

[0070] Multiple complications can result from myelofibrosis. A complication of myelofibrosis can include increased pressure on blood flowing into a patient’s liver. Normally, blood flow from the spleen enters the liver through a large blood vessel called the portal vein. Increased blood flow from an enlarged spleen can lead to high blood pressure in the portal vein (e.g., portal hypertension). This in turn can force excess blood into smaller veins in the stomach and esophagus, potentially causing these veins to rupture and bleed. Pain can be another complication of myelofibrosis. A severely enlarged spleen can cause abdominal pain and back pain. Myelofibrosis can lead to growths in other areas of the body. Myelofibrosis can be associated with bleeding complications. As the disease progresses, platelet count tends to drop below normal (thrombocytopenia) and platelet function becomes impaired. An insufficient number of platelets can lead to easy bleeding. Myelofibrosis can also be associated with painful bones and joints. Myelofibrosis can lead to hardening of bone marrow and inflammation of the connective tissue that is found around the bones. This may cause bone and joint pain. Myelofibrosis can also be associated with development of acute leukemia. Some patients with myelofibrosis develop acute myelogenous leukemia, a type of blood and bone marrow cancer that progresses rapidly.

[0071] In some embodiments of any of the aspects, the subject is a mammal. In some embodiments of any of the aspects, the subject is a human.

[0072] In some embodiments of any of the aspect, the method thereby reduces the amount and / or rate of myelofibrosis in the subject. In some embodiments of any of the aspect, the method thereby reduces the amount of myelofibrosis in the subject. In some embodiments of any of the aspect, the method thereby reduces the rate of myelofibrosis in the subject.

[0073] In some embodiments of any of the aspect, the method thereby reduces the amount and / or rate of myelofibrosis in the spleen of the subject. In some embodiments of any of the aspect, the method thereby reduces the amount of myelofibrosis in the spleen of the subject. In some embodiments of any of the aspect, the method thereby reduces the rate of myelofibrosis in the spleen of the subject.

[0074] In some embodiments of any of the aspect, the method thereby reduces the amount and / or rate of myelofibrosis in the bone marrow of the subject. In some embodiments of any of the aspect, the method thereby reduces the amount of myelofibrosis in the bone marrow of the subject. In some embodiments of any of the aspect, the method thereby reduces the rate of myelofibrosis in the bone marrow of the subject.

[0075] In some embodiments of any of the aspect, the method thereby reduces the amount and / or rate of myelofibrosis in the bone marrow and / or spleen of the subject. In some embodiments of any of the aspect, the method thereby reduces the amount of myelofibrosis in the bone marrow and / or spleen of the subject. In some embodiments of any of the aspect, the method thereby reduces the rate of myelofibrosis in the bone marrow and / or spleen of the subject.

[0076] In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount and / or rate of myelofibrosis in the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount of myelofibrosis in the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the rate of myelofibrosis in the subject.

[0077] In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount and / or rate of myelofibrosis in the spleen of the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount of myelofibrosis in the spleen of the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, andoptionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the rate of myelofibrosis in the spleen of the subject.

[0078] In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount and / or rate of myelofibrosis in the bone marrow of the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount of myelofibrosis in the bone marrow of the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the rate of myelofibrosis in the bone marrow of the subject.

[0079] In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount and / or rate of myelofibrosis in the bone marrow and / or spleen of the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the amount of myelofibrosis in the bone marrow and / or spleen of the subject. In some embodiments of any of the aspect, the amount of a RhoA / ROCK inhibitor, and optionally a JAK inhibitor (e.g., JAK2 inhibitor), administered is an amount sufficient to reduce the rate of myelofibrosis in the bone marrow and / or spleen of the subject.

[0080] In some embodiments of any of the aspects, RhoA / ROCK inhibitors are administered to treat a blood cancer in a subject in need thereof. In some embodiments of any of the aspects, RhoA / ROCK inhibitors are administered to treat an MPN in a subject in need thereof. In some embodiments of any of the aspects, RhoA / ROCK inhibitors are administered to treat primary myelofibrosis or secondary myelofibrosis in a subject in need thereof. In some embodiments of any of the aspects, the subject is in need of treatment for, has, or is diagnosed as having a blood cancer. In some embodiments of any of the aspects, the subject is in need of treatment for, has, or is diagnosed as having an MPN. In some embodiments of any of the aspects, the subject is in need of treatment for, has, or is diagnosed as having primary myelofibrosis or secondary myelofibrosis.

[0081] Subjects having blood cancer, e.g., MPN can be identified by a physician using current methods of diagnosing blood cancer, e.g., MPN. Symptoms and / or complications of blood cancer, e.g., MPN which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, tiredness and shortness of breath, bleeding and bruising easily, enlarged spleen and liver, bone pain, gout, loss of appetite and weight loss, fever, night sweats, itchy skin (pruritus), low number of red blood cells, or low numbers of platelets. Tests that may aid in a diagnosis of blood cancer, e.g. MPN include, but are not limited to, blood tests, bone marrow biopsy, an MRI scan, or a CT scan. A family history of blood cancer, e.g., MPN, or exposure to risk factorsfor blood cancer, e.g., MPN (e.g. exposure to benzene) can also aid in determining if a subject is likely to have blood cancer, e.g., MPN or in making a diagnosis of blood cancer, e.g., MPN.

[0082] The compositions and methods described herein can be administered to a subject having or diagnosed as having blood cancer, e.g., MPN. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. an at least one RhoA / ROCK inhibitor to a subject in order to alleviate a symptom of blood cancer, e.g., MPN. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with the condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.

[0083] The term “effective amount" as used herein refers to the amount of the RhoA / ROCK inhibitor needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the RhoA / ROCK inhibitor that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount" . However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0084] In some embodiments of any of the aspects, an effective amount is an amount that reduces TGFpi secretion from megakaryocytes. In some embodiments of any of the aspects, an effective amount is an amount that reduces TGFp Ire lease from megakaryocytes. In some embodiments of any of the aspects, an effective amount is an amount that reduces the level of TGFpiin a subject’s cell-free blood fraction. In some embodiments of any of the aspects, an effective amount is an amount that reduces the level of TGFpiin a subject’s serum. In some embodiments of any of the aspects, an effective amount is an amount that reduces the size of a subject’s spleen. In some embodiments of any of the aspects, an effective amount is an amount that reduces a subject’s white blood cell count. In some embodiments of any of the aspects, an effective amount is an amount that reduces collagen deposition in a subject’s bone marrow. In some embodiments of any of the aspects, an effective amount is an amount that reduces the TGFpi levels in a subject’s bone marrow. In some embodiments of any of the aspects, an effective amount is an amount that reduces MKclustering. In some embodiments of any of the aspects, an effective amount is an amount that reduces cytokine release. In some embodiments of any of the aspects, an effective amount is an amount that reduces cytokine release from MKs. In some embodiments of any of the aspects, an effective amount is an amount that reduces the amount of fibrosis in a subject’s spleen. In some embodiments of any of the aspects, an effective amount is an amount that reduces the rate of fibrosis in a subject’s spleen. In some embodiments of any of the aspects, an effective amount is an amount that reduces the severity of fibrosis in a subject’s spleen. In some embodiments of any of the aspects, an effective amount is an amount that reduces the amount of fibrosis in a subject’s bones. In some embodiments of any of the aspects, an effective amount is an amount that reduces the rate of fibrosis in a subject’s bones. In some embodiments of any of the aspects, an effective amount is an amount that reduces the severity of fibrosis in a subject’s bones. In some embodiments of any of the aspects, an effective amount is an amount that reduces the amount of fibrosis in a subject’s bone marrow. In some embodiments of any of the aspects, an effective amount is an amount that reduces the rate of fibrosis in a subject’s bone marrow. In some embodiments of any of the aspects, an effective amount is an amount that reduces the severity of fibrosis in a subject’s bone marrow.

[0085] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the at least one RhoA / ROCK inhibitor, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for platelet levels, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0086] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising the at least one RhoA / ROCK inhibitor as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise the at least one RhoA / ROCK inhibitor as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of the RhoA / ROCK inhibitor as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of the RhoA / ROCK inhibitor as described herein.Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some nonlimiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. the at least one RhoA / ROCK inhibitor as described herein.

[0087] In some embodiments, the pharmaceutical composition comprising the at least one RhoA / ROCK inhibitor as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®- type dosage forms and dose-dumping.

[0088] Suitable vehicles that can be used to provide parenteral dosage forms of the at least one RhoA / ROCK inhibitor as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of an agent as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0089] Pharmaceutical compositions comprising the at least one RhoA / ROCK inhibitor can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil- in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0090] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the at least one RhoA / ROCK inhibitor can be administered in a sustained release formulation.

[0091] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0092] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0093] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.

[0094] In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor described herein is administered as a monotherapy, e.g., another treatment for the blood cancer is not administered to the subject.

[0095] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and / or treatment can include blood transfusion, platelet transfusion, corticosteroids, immune suppressants, splenectomy, and plasma exchange. In some embodiments of any of the aspects, the second agent and / or treatment to the subject is a JAK2 inhibitor.

[0096] In some embodiments of any of the aspects, the combinatorial therapy comprises administering the at least one RhoA / ROCK inhibitor and the at least one JAK2 inhibitor. In some embodiments of any of the aspects, the combinatorial therapy comprises administering the at least one RhoA / ROCK inhibitor and the at least one JAK2 inhibitor sequentially and / or separately. In some embodiments of any of the aspects, the combinatorial therapy comprises administering the at least one RhoA / ROCK inhibitor and the at least one JAK2 inhibitor contemporaneously.

[0097] In certain embodiments, an effective dose of a composition comprising the at least one RhoA / ROCK inhibitor as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising the at least one RhoA / ROCK inhibitor can be administered to a patient repeatedly. For systemic administration, subjects can be administereda therapeutic amount of a composition comprising the at least one RhoA / ROCK inhibitor, such as, e.g. 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0098] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more. In some embodiments of any of the aspects, the methods described herein increase platelet levels in the subject.

[0099] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient(s). The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising the at least one RhoA / ROCK inhibitor can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0100] The dosage ranges for the administration of the at least one RhoA / ROCK inhibitor, according to the methods described herein depend upon, for example, the form of the at least one RhoA / ROCK inhibitor, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage to which, for example, amount and / or rate of myelofibrosis and / or cytokine release is desired to be decreased. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0101] The efficacy of the at least one RhoA / ROCK inhibitor in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. decreasing amount and / or rate ofmyelofibrosis) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. myelofibrosis levels. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, forthat disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. myelofibrosis and / or pro-fibrotic cytokine release levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of a murine model of blood cancer, e.g., MPN. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. myelofibrosis levels.

[0102] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not (“comprising”). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0103] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0104] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0105] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0106] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0107] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0108] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of blood cancer, e.g., MPN. A subject can be male or female.

[0109] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. blood cancer, such as MPN) or one or more complications related to such a condition, and optionally, have already undergone treatment for blood cancer, e.g., MPN or the one or more complications related to blood cancer, e.g., MPN Alternatively, a subject can also be one who has not been previously diagnosed as having blood cancer, e.g., MPN or one or more complications related to blood cancer, e.g., MPN. For example, a subject can be one who exhibits one or more risk factors for blood cancer, e.g., MPN or one or more complications related to blood cancer, e.g., MPN, or a subject who does not exhibit risk factors.

[0110] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0111] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0112] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, thenucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0113] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0114] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0115] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0116] In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor described herein is exogenous. In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor described herein is ectopic. In some embodiments of any of the aspects, the at least one RhoA / ROCK inhibitor described herein is not endogenous.

[0117] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found inan unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0118] In some embodiments, a nucleic acid as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0119] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0120] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0121] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0122] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0123] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0124] In some embodiments of any of the aspects, an inhibitor is an inhibitory nucleic acid. In some embodiments of any of the aspects, inhibitors of the expression of a given gene can be an inhibitory nucleic acid. As used herein, “inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double -stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like. In some embodiments of any of the aspects, the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA). Inhibitory nucleic acids can also include guide sequence molecules (e.g., a guide RNA) that function, e.g., in combination with an enzyme, to induce insertions, deletions, indels, and / or mutations of a target, thereby inhibiting the expression of the target.

[0125] In some embodiments of any of the aspects, an iNA comprises a sequence that is complementary to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 15 nucleotides in length that is complementary to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 20 nucleotides in length that is complementary to at least a portion of a target sequence described herein.

[0126] In some embodiments of any of the aspects, an iNA comprises a sequence that is the reverse complement to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 15 nucleotides in length that is the reverse complement to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 20 nucleotides in length that is the reverse complement to at least a portion of a target sequence described herein.

[0127] In some embodiments of any of the aspects, an iNA comprises a sequence that can specifically hybridize to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 15 nucleotides in length that can specifically hybridize to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 20 nucleotides in length that can specifically hybridize to at least a portion of a target sequence described herein.

[0128] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and / or activity of the target.

[0129] As used herein, the term “iRNA” refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In some embodiments of any of the aspects, an iRNA as described herein effects inhibition of the expression and / or activity of a target, e.g. RhoA / ROCK. In some embodiments of any of the aspects, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA. In some embodiments of any of the aspects, administering an inhibitor (e.g. an iRNA) to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.

[0130] In some embodiments of any of the aspects, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron boundaries. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0131] Exemplary embodiments of types of inhibitory nucleic acids can include, e.g,. siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art. One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of, e.g., RhoA / ROCK, e.g., using publically available design tools. siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).

[0132] In some embodiments of the various aspects described herein, the inhibitory nucleic acid is a guide nucleic acid (gNA). As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide RNA,” “single guide RNA,” “gRNA” or “CRISPR guide sequence” refer to a nucleic acid comprising a sequence that determines the specificity of an enzyme, e.g., the Cas DNA binding protein of a CRISPR / Cas system, to a polynucleotide target. The gNA can comprise a polynucleotide sequence with at least partial complementarity with a target nucleic acid sequence, sufficient to hybridize with the target nucleic acid sequence and to direct sequence -specific binding of an enzyme, e.g, a nuclease, to the target nucleic acid sequence.

[0133] In some embodiments, the enzyme directed by the gNA is a gene-editing protein, e.g., any nuclease that induces a nick or double-strand break into a desired recognition site. Such enzymes can be native or engineered. These breaks can then be repaired by the cell in one of two ways: non- homologous end joining and homology-directed repair (homologous recombination). In non- homologous end joining (NHEJ), the double-strand breaks are repaired by direct ligation of the break ends to one another. As such, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion. In homology-directed repair, a donor polynucleotide with homology to the cleaved target DNA sequence can be used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material may be inserted / copiedinto the site. The modifications of the target DNA due to NHEJ and / or homology-directed repair can be used for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

[0134] In some embodiments, the gene-editing protein is a CRISPR-associated nuclease. The native prokaryotic CRISPR-associated nuclease system comprises an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats), and CRISPR-associated ("Cas") nuclease proteins. The RNA of the transcribed CRISPR array is processed by a subset of the Cas proteins into small guide RNAs, which generally have two components as discussed below. There are at least three different systems: Type I, Type II and Type III. The enzymes involved in the processing of the RNA into mature crRNA are different in the 3 systems. In the native prokaryotic system, the guide RNA ("gRNA") comprises two short, non-coding RNA species referred to as CRISPR RNA ("crRNA") and transacting RNA ("tracrRNA"). In an exemplary system, the gRNA forms a complex with a nuclease, for example, a Cas nuclease. The gRNA: nuclease complex binds a target polynucleotide sequence having a protospacer adjacent motif ("PAM") and a protospacer, which is a sequence complementary to a portion of the gRNA. The recognition and binding of the target polynucleotide by the gRNA: nuclease complex induces cleavage of the target.

[0135] Any CRISPR-associated nuclease can be used in the system and methods of the invention. CRISPR nuclease systems are known to those of skill in the art, e.g. Cas9, Cas 12, Cas 12a, or the like, see Patents / applications 8,993,233, US 2015 / 0291965, US 2016 / 0175462, US 2015 / 0020223, US 2014 / 0179770, 8,697,359; 8,771,945; 8, 795,965; WO 2015 / 191693; US 8,889,418; WO 2015 / 089351; WO 2015 / 089486; WO 2016 / 028682; WO 2016 / 049258; WO 2016 / 094867; WO 2016 / 094872; WO 2016 / 094874; WO 2016 / 112242; US 2016 / 0153004; US 2015 / 0056705; US 2016 / 0090607; US 2016 / 0029604; 8,865,406; 8,871,445; each of which are incorporated by reference in their entirety. The nuclease can also be a phage Cas nuclease, e.g., Cas<b (e.g., Pausch et al. Science 369:333-7 (2020); which is incorporated by reference herein in its entirety).

[0136] The full-length guide nucleic acid strand can be any length. For example, the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments of the various aspects described herein, a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. For example, the guide nucleic acid sequence is 10-30 nucleotides long.

[0137] In addition to a sequence that is complementary to a target nucleic acid, in some embodiments, the gNA also comprises a scaffold sequence. Expression of a gNA encoding both a sequence complementary to a target nucleic acid and scaffold sequence has the dual function of bothbinding (hybridizing) to the target nucleic acid and recruiting the endonuclease to the target nucleic acid, which may result in site-specific CRISPR activity. In some embodiments, such a chimeric gNA may be referred to as a single guide RNA (sgRNA).

[0138] In some embodiments of the various aspects described herein, the guide nucleic acid is designed using a guide design tool (e.g., Benchling™; Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™; E-CRISP™; Geneious™; GenHub™; GUIDES™ (e g., for library design); Horizon Discovery™; IDT™; Off-Spotter™; and Synthego™; which are available on the world wide web).

[0139] In some embodiments of any of the aspects, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments of any of the aspects, the modified RNA will have a phosphorus atom in its intemucleoside backbone.

[0140] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Modified RNA backbones that do not include a phosphoms atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or oneor more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular — CH2— NH— CH2— , —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], — CH2— O— N(CH3)— CH2— , — CH2— N(CH3)— N(CH3)- -CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as — O-P-O-CH2-],

[0141] In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0142] The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833- 843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0143] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or agroup for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments of any of the aspects, the modification includes a 2' methoxyethoxy (2'-O— CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-M0E) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'- DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O— CH2— O— CH2— N(CH2)2, also described in examples herein below.

[0144] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'- 5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0145] An inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5- halo, particularly 5 -bromo, 5 -trifluoromethyl and other 5 -substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 8 -azaguanine and 8 -azaadenine, 7-deazaguanine and 7- daazaadenine and 3 -deazaguanine and 3 -deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O- methoxy ethyl sugar modifications.

[0146] The preparation of the modified nucleic acids, backbones, and nucleobases described above are well known in the art.

[0147] Another modification of an inhibitory nucleic acid featured in the invention involves chemically linking to the inhibitory nucleic acid to one or more ligands, moieties or conjugates thatenhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-O-hexadecyl-rac-glycero-3 -phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Then, 1996, 277:923-937).

[0148] Antibody reagents specific for the targets and / or markers described herein, e.g., RhoA / ROCK are known in the art. For example, such reagents are readily commercially available, e.g., the anti-RhoA antibodies 1A11-4G10 (Invitrogen, Cat. No. MAI-011), 1B8-1C7 (Invitrogen, Cat. No. MAI-134), 1B3-4A10 (Invitrogen, Cat. No. MAI-123), 1B3D7 (Invitrogen Cat. No. 66733- 1-IG), EPR18134 (Abeam, Cat. No. abl87027), EPR18133 (Abeam Cat. No. abl87026), and 1B12 (Abeam, Cat. No. ab54835). For example, such reagents are readily commercially available, e.g., the anti-ROCKl antibodies GT261 (Invitrogen, Cat. No. MA5-27778), GT464 (Invitrogen, Cat. No. MA5-27779), EPR638Y (Abeam, Cat. No. ab!34181), and EP786Y (Abeam, Cat. No. ab45171). ). For example, such reagents are readily commercially available, e.g., the anti-ROCKl antibodies 1C7B8 (Invitrogen, Cat. No. 66633-1-IG), ARC0744 (Invitrogen, Cat. No. MA5-35356), and EPR7141(B) (Abeam, Cat. No. abl25025).

[0149] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. blood cancer such as MPN. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with blood cancer, e.g., MPN. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one ormore symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0150] In some embodiments of any of the aspects, treatment is measured by myelofibrosis levels, e.g, treatment occurs if the subject’s myelofibrosis levels decrease. In some embodiments of any of the aspects, treatment is measured by cytokine release levels, e.g, treatment occurs if cytokine release by MKs decreases.

[0151] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to uncontrolled bleeding, surgery, need for a transfusion, or internal bleeding. In some embodiments, the methods described herein can be prophylactic relative to myelofibrosis. In some embodiments, the methods described herein can be prophylactic relative to decreases in mature blood cell levels. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0152] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0153] As used herein, the term “nanoparticle” refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle.Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles. Lipid nanoparticles can comprise multiple componenents, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.

[0154] Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.

[0155] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0156] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0157] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0158] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0159] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0160] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0161] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0162] As used herein, the term “specific binding” refers to a chemical interaction between twomolecules, compounds, cells and / or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[0163] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0164] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0165] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0166] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0167] Other terms are defined herein within the description of the various aspects of the invention.

[0168] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0169] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes canbe made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0170] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0171] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method of treating a blood cell cancer in a subject in need thereof, the method comprising administering to the subject a RhoA / ROCK inhibitor.2. The method of paragraph 1, wherein the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; RKI-1447; Y-27632, GSK429286A; Y-30141; AT- 13148; BA-210; [3-elemene; belumosudil; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; ibuprofen; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.3. The method of paragraph 2, wherein the RhoA / ROCK inhibitor is belumosudil, netarsudil, ripasudil, or fasudil.4. The method of paragraph 2, wherein the RhoA / ROCK inhibitor is belumosudil or netarsudil.5. The method of any of the preceding paragraphs, wherein the subject has or is diagnosed as having a myeloproliferative neoplasm.6. The method of any of the preceding paragraphs, wherein the subject has or is diagnosed as having primary myelofibrosis or secondary myelofibrosis.7. The method of any of the preceding paragraphs, wherein the method further comprises administering to the subject a JAK2 inhibitor.8. The method of paragraph 7, wherein the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; filgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868.9. The method of paragraph 7, wherein the JAK2 inhibitor is ruxolitinib.10. The method of any one of the preceding paragraphs, wherein the subject is a mammal.11. The method of any one of the preceding paragraphs, wherein the subject is human.12. The method of any one of the preceding paragraphs, whereby the amount and / or rate of myelofibrosis in the subject is reduced.13. The method of any one of the preceding paragraphs, whereby the amount and / or rate of myelofibrosis in the bone marrow and / or spleen of the subject is reduced.

[0172] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method of treating a blood cancer in a subject in need thereof, the method comprising administering to the subject a RhoA / ROCK inhibitor.2. The method of paragraph 1, wherein the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; belumosudil;RKI-1447; Y-27632; GSK429286A; Y- 30141; AT-13148; BA-210; [3-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.3. The method of paragraph 1, wherein the RhoA / ROCK inhibitor is selected from the group consisting of:Netarsudil; Y-30141; BA-210; [3-elemene; GSK-576371; H-1152; Y-33075;Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001;GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B;AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677;LX-7101; GSK180736; Y-27632; Y-27632 dihydrochloride; Fasudil; TS-f22;Hydroxyfasudil; Belumosudil; and NRL-1049.4. The method of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor is a ROCK1 inhibitor.5. The method of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor is a ROCK1 / 2 inhibitor.6. The method of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is selected from the group consisting of: chroman 1; BAY-549; TCS-7001; GSK269962A; verosudil; Y 33983; AMA-0076;DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622GSK429286A;RKI-1447; ripasudil; SR3677; GSK180736; and Y-27632.7. The method of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is Y-27632.8. The method of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 300 nM or lower.The method of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor has an IC50 for R0CK1 of 250 nM or lower. The method of any one of the preceding paragraphs, wherein the subject has or is diagnosed as having a myeloproliferative neoplasm. The method of any one of the preceding paragraphs, wherein the subject has or is diagnosed as having aberrant megakaryopoiesis, essential thrombocythemia, or polycythemia vera. The method of any one of the preceding paragraphs, wherein the subject has or is diagnosed as having Myelofibrosis (MF). The method of any one of the preceding paragraphs, wherein the subject has or is diagnosed as having primary myelofibrosis or secondary myelofibrosis. The method of any one of the preceding paragraphs, wherein the method further comprises administering to the subject a JAK2 inhibitor. The method of paragraph 14, wherein the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; filgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868. The method of paragraph 14, wherein the JAK2 inhibitor is ruxolitinib. The method of any one of the preceding paragraphs, wherein the subject is a mammal. The method of any one of the preceding paragraphs, wherein the subject is human. The method of any one of the preceding paragraphs, whereby the amount and / or rate of myelofibrosis in the subject is reduced. The method of any one of the preceding paragraphs, whereby the amount and / or rate of myelofibrosis in the bone marrow and / or spleen of the subject is reduced. A RhoA / ROCK inhibitor, and optionally a JAK2 inhibitor, for use in the treatment of blood cancer. The inhibitor of paragraph 21, wherein the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; belumosudil;RKI-1447; Y-27632; GSK429286A; Y- 30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983. The inhibitor of paragraph 21, wherein the RhoA / ROCK inhibitor is selected from the group consisting of:Netarsudil; Y-30141; BA-210; p-elemene; GSK-576371; H-1152; Y-33075;Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001;GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B;AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677;LX-7101; GSK180736; Y-27632; Y-27632 dihydrochloride; Fasudil; TS-f22;Hydroxyfasudil; Belumosudil; and NRL-1049.24. The inhibitor of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor is a R0CK1 inhibitor.25. The inhibitor of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor is a R0CK1 / 2 inhibitor.26. The inhibitor of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor, R0CK1 inhibitor, or R0CK1 / 2 inhibitor is selected from the group consisting of: chroman 1; BAY-549; TCS-7001; GSK269962A; verosudil; Y 33983; AMA-0076;DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622GSK429286A;RKI-1447; ripasudil; SR3677; GSK180736; and Y-27632.27. The inhibitor of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor, R0CK1 inhibitor, or R0CK1 / 2 inhibitor is Y-27632.28. The inhibitor of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor has an IC50 for R0CK1 of 300 nM or lower.29. The inhibitor of any one of the preceding paragraphs, wherein the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 250 nM or lower.30. The inhibitor of any one of the preceding paragraphs, the blood cancer is a myeloproliferative neoplasm.31. The inhibitor of any one of the preceding paragraphs, wherein the subject has or is diagnosed as having aberrant megakaryopoiesis, essential thrombocythemia, or polycythemia vera.32. The inhibitor of any one of the preceding paragraphs, the blood cancer is Myelofibrosis (MF).33. The inhibitor of any one of the preceding paragraphs, the blood cancer is primary myelofibrosis or secondary myelofibrosis.34. The inhibitor of any one of the preceding paragraphs, wherein the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; filgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868.35. The inhibitor of any one of the preceding paragraphs, wherein the JAK2 inhibitor is ruxolitinib.

[0173] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0174] Described herein are methods of targeting the RhoA / ROCK pathway to manipulate autophagosome formation and manipulate the release of cytokines from hematopoietic cells in the bone marrow to protect from myelofibrosis. Rho / ROCK inhibition can reduce fibrosis, normalize TGFpi levels, and protect against myelofibrosis in patients with blood cell cancer (e.g. myeloproliferative neoplasms).

[0175] Megakaryocytes are large, polyploid platelet precursor cells that primarily reside within the bone marrow. Recent advances in single-cell omics have identified that in addition to platelet production, MKs contribute to immunity as well as to the maintenance of bone marrow homeostasis, functions that might be hijacked under disease circumstances.

[0176] In patients with primary and secondary myelofibrosis, subsets of myeloproliferative neoplasms that are instigated by mutations in the thrombopoietin receptor MPL or its downstream effector Janus kinase (JAK) 2, immature MKs accumulate in the bone marrow. These aberrantly matured MKs release pro-fibrotic cytokines such as transforming growth factor P (TGFpi) and interleukin (IL) ip into the bone marrow, where they induce fibrotic alterations that ultimately cause pancytopenia and represent the major cause of mortality in patients with myelofibrosis. While current treatment aims at preventing megakaryocytosis and MK differentiation defects in myelofibrosis, directly targeting cytokine release from MKs has not yet been explored. Therefore, it was hypothesized that decreasing aberrant cytokine release from profibrotic MKs by targeting RhoA / autophagy pathways could ameliorate myelofibrosis progression in vivo.

[0177] To investigate this, murine MKs were cultured and co-localization of TGFpi with different a-granule proteins were analyzed by super-resolution confocal microscopy. In contrast to previous observations, this data revealed a distinct localization pattern of TGFp 1 independent of a- granule cargo, suggesting a distinct release machinery. Hematopoietic stem and progenitor cells were next treated with inhibitors of RhoA and autophagy signaling pathways and MK differentiation and TGFpi secretion were assessed. Treatment of MKs with autophagy or RhoA / ROCK inhibitors markedly reduced TGFpi secretion in vitro and was associated with an intracellular accumulation of TGFpi. In addition, TGFpi strongly colocalized with the autophagy marker light chain (LC) 3B in native murine MKs isolated from the bone marrow by size filtration.

[0178] To establish whether the examined signaling pathways were altered in vivo, two preclinical models of myelofibrosis were utilized, an MPLW515L-driven transplant model and mice lacking the inhibitory MK receptor G6b-B (Mpig6b~ / ~). Ex vivo, basal autophagy and RhoA levels were increased in platelets derived from mice with MPLW515L-driven myelofibrosis, suggesting aberrant activity of both pathways upon disease progression. Next, it was tested whether lack of theautophagy protein Atg5 from hematopoietic stem cells (AtgJl / ^l,MxCre) might affect disease progression in the MPLW515L-driven myelofibrosis model. Intriguingly, lack of Atg5 prevented leukocytosis, MK clustering and aberrant TGFpi secretion, suggesting beneficial effects of autophagy inhibition on disease progression beyond cytokine secretion. Lastly, it was investigated whether treatment of MPLW515L- transplanted mice with a ROCK inhibitor would affect fibrosis and reduced fibrosis in spleens and femurs of treated mice was found. This is of specific interest, since several ROCK inhibitors are already FDA-approved, i.e., for the treatment of graft-versus-host-disease. In line with these findings, Mpig6b~ mice with an additional MK- and platelet-selective deficiency in RhoA (RhoA^,p^4Cre) displayed normalized TGFpi levels within the bone marrow and were protected from myelofibrosis. Selective inhibitors are currently further being tested in vivo in combination with the JAK2 inhibitor ruxolitinib.

[0179] The effectiveness of RhoA / ROCK inhibitors for the prevention of fibrotic alterations in myelofibrosis is demonstrated in vivo findings showing potential for ameliorated disease upon ROCK inhibition or RhoA deficiency.Example 2

[0180] Cytokine secretion from megakaryocytes is governed by small GTPases through autophagy

[0181] Megakaryocytes (MKs) are large cells primarily residing in the bone marrow. While it was previously assumed that the sole function of MKs is to produce platelets, recent studies using single cell RNA sequencing have revealed novel subsets of MKs that serve immune functions and maintain stem cell quiescence through the secretion of cytokines such as platelet factor 4 (PF4) and transforming growth factor pi (TGFpi). However, while the secretion of these bioactive molecules from platelets requires activation, little is known about what triggers cytokine release from MKs in health and disease. During neoplastic transformation, e.g. upon mutation of the thrombopoietin receptor MPL or its downstream effector kinase JAK2, immature MKs accumulate in the bone marrow, where they release high levels of PF4 and TGFp 1 ultimately causing myelofibrosis and pancytopenia. While current treatment strategies for myeloproliferative neoplasms aim to prevent MK hyperproliferation, an understanding of why cytokine secretion is dysregulated in neoplastic MKs and whether it can be specifically targeted is lacking.

[0182] Previous studies have suggested TGFpi secretion from fibroblasts occurs via secretory autophagy, the release rather than degradation of autophagosomes. The inventors hypothesized that upregulation of autophagy in MKs might promote enhanced TGFpi release during myelofibrosis. Using an MPLW515L-driven transplant model of myelofibrosis, increased levels of the autophagosome marker light chain (LC) 3B was found in platelets from diseased mice. In line withthis, it was identified that TGFp 1 strongly co-localized with the autophagy marker light chain (LC) 3B in native MKs. The GTPase RhoA and its downstream effector Rho kinase 1 (R0CK1) are major regulators of basal autophagy in fibroblasts and upregulated in MKs in myelofibrosis, while loss of the GTPase dynamin 2 from MKs was previously demonstrated to induce bone marrow fibrosis due to increased TGFpi release, overall suggesting an important role of small GTPases in regulating cytokine secretion from MKs. To test this hypothesis in vitro, cultured MKs were treated with inhibitors of RhoA or R0CK1, which reduced TGFpi secretion and caused an intracellular accumulation of both TGFpi as well as the autophagy marker LC3B. In contrast, MKs treated with a small molecule inhibitor of the dynamin 2-associated GTPase Arf6 exhibited reduced intracellular TGFpi and LC3B suggesting opposing roles of the GTPases RhoA and Arf6 in governing TGFpi secretion.

[0183] To investigate this regulation in vivo, the bone marrow of mice with a selective deletion of Arf6 in MKs and platelets (Arf6fl / fl,Pf4Cre) was analyzed. Arf6fl / fl,Pf4Cre exhibited an expansion of their hematopoietic stem and progenitor cell compartment as well as a 2-fold increase in MK numbers. In line with dynamin 2-deficient mice, which develop progressive bone marrow fibrosis, Arf6fl / fl,Pf4Cre mice displayed increased levels of TGFp 1 in their bone marrow fluid, which was accompanied by collagen deposition in the bone marrow. Together with the in vitro data, these findings suggest an important role of Arf6 as a negative regulator of autophagy-induced TGFpi secretion.

[0184] To investigate whether inhibition of Rho A / ROCK1 -induced secretory autophagy in MKs on the other hand might ameliorate myelofibrosis pathogenesis, mice with a selective deletion of Rhoa in MKs were generated and the MPLW515L-driven transplant model was utilized to induce disease. Recipient mice transplanted with MPLW515L-transduced RhoA-deficient cells displayed reduced MK clustering and collagen deposition in the bone marrow. Moreover, treatment of MPLW515L- transplanted mice with the JAK2 inhibitor ruxolitinib in combination with the ROCK inhibitor Y27632 significantly reduced spleen size and collagen deposition beyond ruxolitinib treatment alone, thus attenuating disease hallmarks. Most importantly, only combination treatment with Y27632 significantly reduced TGFpi levels in the bone marrow strongly suggesting secretory autophagy as its main secretion pathway.

[0185] In summary, this data indicates that TGFpi secretion from MKs is regulated by small GTPases through secretory autophagy, a targetable pathway with therapeutic potential for the treatment of myelofibrosis.

[0186] Example 3

[0187] Megakciryo- and thrombopoiesis

[0188] Platelets are small anucleate cell fragments mainly responsible for maintaining vascular integrity and hemostasis upon vessel damage. Platelet numbers of 150 - 400 x 103per pL in humans are maintained by large precursor cells, called megakaryocytes (MKs), that reside in the bone marrow (BM). To generate platelets, MKs breach the endothelial lining and release long cytoplasmic protrusions into sinusoids.1MKs derive from hematopoietic stem cells (HSCs), which, due to their lifelong self-renewal potential, can give rise to numerous progenitor cells with increasing lineage specification. MKs are the largest cells within the BM but only account for 0.05 - 0. 1% of the whole BM population.2In addition to their essential function as platelet progenitors, they also modulate the BM microenvironment by directly affecting HSC quiescence3,4or enabling osteoblast expansion after irradiation.5MK maturation is a strictly organized process and highly dependent on precise transcriptional activation and repression induced by a variety of cytokines, most prominently thrombopoietin (TPO). TPO-deficient mice exhibited significantly reduced HSC numbers in addition to severe thrombocytopenia (platelet counts below 10% compared to control animals),6presumably due to a reduced secretion of platelet factor 4 (Pf4 / CXCL4) from mature MKs.7TPO binding to its receptor myeloproliferative leukemia protein (c-Mpl) induces an intracellular signaling cascade culminating in the transcription of MK-specific genes and transcription factors.8MK maturation is characterized by an expansion of specific granules as well as the development of an elaborate membrane system, the demarcation membrane system (DMS).9Two major granule types are assembled within MKs: a- and dense granules. A plethora of pro- and anti -angiogenic molecules, growth factors, chemotactic agents and coagulation factors are stored in the most abundant granule subtype, a-granules (50-80 per platelet), while dense granules (3-8 per platelet) contain small cationic molecules and nucleotides such as calcium, adenosine diphosphate (ADP), ATP and serotonin.10Moreover, megakaryopoiesis encompasses endomitosis, a polyploidization involving cycles of DNA replication without cell division. These unique processes of cytoplasmic and DNA maturation are indispensable to ultimately enable guided proplatelet formation into vessel sinusoids.

[0189] The main function of mature MKs is the generation of platelets, which are released from the tip of a nascent proplatelet and further mature into platelets within the vasculature. Defects in both actin or microtubule cytoskeletal rearrangements impair platelet production, culminating in thrombocytopenia.11Filamentous (F-)actin is further indispensable for the generation of a functional DMS, since it transduces signals downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) and thus enables membrane invaginations.9Small GTPases of the Rho family are essential for F-actin rearrangements. While the GTPases Cdc42 and Rael are critical regulators of actin and microtubule dynamics during proplatelet formation, RhoA is markedly involved in platelet granule secretion and stress fiber formation.12,13Small GTPases of the Rab and Arf family on the other hand have been described to contribute to platelet membrane dynamics, most importantly to endo- and exocytictrafficking of granules by interacting with proteins of the soluble NSF attachment protein receptors (SNARE) family, e.g. vesicle -associated membrane protein-3 (Vamp3).14 15

[0190] MK and platelet secretion in health and disease. Upon stimulation with agonists platelets become activated and release their granular contents. This secretion property allows platelets to function as a major transport and rapid delivery system in the blood. Defective a-granule synthesis in patients with disease-causing variants in the NBEAL2 gene encoding for the BEACH-domain containing neurobeachin 2 leads to grey platelet syndrome (GPS), a severe bleeding diathesis.16Comparable defects in platelet function causing impaired hemostasis due to hampered a-granule cargo retention are also observed in Nbeal2-deficient mice.1718The mice further exhibit fibrotic alterations within the BM cavity, a disease termed myelofibrosis, presumably due to an aberrant release of cytokines like the profibrotic transforming growth factor [31 (TGF[31) from MKs. This has been observed in several other mouse models with defective a-granule biosynthesis (Becker et al., unpublished;19,20) and suggests that cytokine release is independent of directed granule secretion, however, the underlying signaling mechanisms remain elusive.

[0191] The release of a-granules is a strictly coordinated process facilitated by membrane- associated SNARE family proteins, which are indispensable for membrane docking of vesicles. Genetic manipulation of the major SNAREs involved in platelet granule secretion in mice revealed that a substantial reduction in secretion (> 70%) resulted in protection from occlusive thrombus formation in vivo.27This study, however, focused on the importance of granule secretion on platelet function, but did not shed light on how disruption of SNAREs affected the release of granules from MKs.

[0192] Intriguingly, MKs are long thought to not only maintain physiological platelet counts, but to also be indispensable for BM homeostasis and HSC self-renewal. MK-released TGFJ31 as well as Pf4 was identified to be critically involved in maintaining HSC quiescence3,4MKs also directly stimulate osteoblast proliferation in vitro as well as in vivo upon irradiation and interfere with osteoclast differentiation suggesting a coordinated release of cytokines and growth factors into the BM (Figure 1).22,23,25,28

[0193] Among all of the cytokines released by MKs, TGFJ31 appears to be of particular significance, not only for osteogenesis,24but also for the pathophysiology of myeloproliferative diseases. Among these, primary myelofibrosis (PMF) in humans is characterized by an enhanced deposition of collagen and reticulin fibers in the BM and spleen leading to impaired hematopoiesis and defective blood cell generation.29Disease-causing variants in the genes encoding for janus kinase 2 (JAK2), the TPO receptor c-MPL or calreticulin (CALR) are the most common reasons for the development of myelofibrosis by inducing constitutive activation of signaling pathways important for hematopoiesis.30PMF clusters are characterized by an accumulation of immature MKs, which are thought to exhibit an increased release of profibrotic cytokines. Of these, TGFJ31 (Figure 1) has notonly been shown to inhibit matrix metalloproteinase (MMP) activity, but to also increase collagen and proteoglycan synthesis in MK-adjacent cells.29Interestingly, inhibition of TGFJ31 activity using a protein trap significantly ameliorated myelofibrosis progression in a mouse model of myelofibrosis by inhibiting TGF[31 signaling in a paracrine and autocrine manner thus highlighting its prominent role in myelofibrosis progression.31

[0194] The autophagic machinery in unconventional protein secretion. It remains unclear whether the release of TGFp 1 is driven by a directed secretion of granules versus a leakage of excess protein due to impaired granule packaging within defective MKs. In the work described herein, it was determined if MKs utilize similar secretion mechanisms.

[0195] Under normal physiology, autophagy is induced upon nutrient starvation and enables the recycling and degradation of aged / damaged organelles or aggregation-prone proteins as well as other cellular components through the fusion of autophagosomes with lysosomes.33,34Classic autophagy is induced, when the energy-sensing kinases mechanistic target of rapamycin complex (mTOR) and AMP-activated protein kinase (AMPK) activate Unc-51-like autophagy activating kinase 1 (ULK1), which translocates to the endoplasmic reticulum (ER) to induce the formation of a phagophore termed omegasome.35Local production of PI3 phosphate through the PI3 kinase Vps34 promotes lipidation of the main ULK1 effector microtubule-associated protein light chain 3 isoform B (LC3B), which is a member of the autophagy-related protein (Atg) 8 family and drives omegasome expansion (Figure 2).35In addition to Atg8 proteins, autophagosome formation requires the sequential spatial and temporal activity of several other Atg proteins,36among which Atg9 is essential for autophagosome formation by supplying key components to autophagosomal membranes,37while Atg7 and Atg3 serve as El and E2 enzymes.38Interestingly, lack of Atg7 from HSCs results in markedly impaired megakaryo- and thrombopoiesis, whereas a platelet and MK-specific deletion of the protein only impaired hemostasis and thrombosis, while MK maturation appeared unaffected.39,40

[0196] How deletion of Atg proteins affects MK secretion and thus influences maintenance of the hematopoietic niche, however, remained uninvestigated. A comparable defect in megakaryopoiesis was observed in vitro upon treatment of BM- or fetal liver-derived progenitors with the autophagy-inducing drug rapamycin that targets mTOR and leads to impaired polyploidization and cell growth.41Similarly, interference with late autophagy using the vacuolar H+ -ATPase inhibitor bafilomycin Al diminished MK maturation in vitro,42however, both rapamycin and bafilomycin Al are broad range drugs and exhibit a plethora of off-target effects on cell proliferation and are thus not ideal to specifically target autophagy.43

[0197] In addition to canonical autophagy that eventually culminates in lysosomal degradation of cargo, recent advances have identified unconventional trafficking and release of proteins involving the autophagic machinery, termed secretory autophagy.44The first evidence for alternative secretory pathways came from the identification of purely cytosolic proteins lacking an N-terminal ER signalpeptide, which were nonetheless secreted from cells,45among them the yeast-derived acyl coenzyme A-binding protein (AcbA) 46 as well as macrophage-derived interleukin (IL) ip.47Of note, similarly to TGF i, IL 1 p is assembled as an inactive precursor in the cytosol and is only cleaved into its active form upon secretion.48Comparable to autophagy-independent unconventional secretion both in yeast and mammalian cells,32,49secretory autophagy also relies on Golgi reassembly stacking proteins (GRASPs) and the endosomal sorting complex required for transport (ESCRT) that is indispensable for multivesicular body (MVB) formation.44The involvement of endosomal proteins in unconventional cytokine release was endorsed by in vitro findings, identifying a role of the small G protein ADP ribosylation factor 6 (Arf6) in autophagosome formation by regulating PIP2 levels in the plasma membrane.50While Arf6 has previously been shown to be important for vesicular trafficking of platelet-specific allb 3 integrins,51its role in unconventional cytokine secretion by MKs has yet to be explored. The finding of an Arf6- dependent autophagosome formation is especially intriguing, since Arf6 is activated by the large GTPase dynamin 2, which is critical for endosomal vesicle transport (Figure 2).52Strikingly, MK- and platelet-specific deficiency in dynamin 2 is associated with a progressive myelofibrosis in mice due to increased TGFpi release, thus suggesting altered secretory pathways to play a role therein.53

[0198] While the mechanisms of platelet secretion have been extensively studied, whether MKs undergo physiological secretion, how the release may be regulated, and to what degree it becomes dysregulated under disease conditions is not well understood and will be addressed in the project described herein.

[0199] Initial Work. A mouse line with a spontaneous mutation leading to severe macrothrombocytopenia and myelofibrosis was characterized. A single nucleotide exchange was identified that lead to loss of protein expression in a splice acceptor site of the gene encoding for megakaryocyte and platelet inhibitory receptor G6b-B (Mpig6b), an immunoreceptor tyrosine-based inhibition motif (ITIM) receptor that was previously described to be responsible for the regulation of platelet reactivity and production.54,55It was found that G6b-B, in addition to regulating platelet counts and reactivity (Figure 3A, 3B), is essential in promoting the activation of MK- specific gene transcription by increasing TPO signaling responsiveness. Using a series of in vitro and ex vivo analyses, it was uncovered that MKs in Mpig6b-mutant MKs were significantly smaller in size, exhibited reduced ploidy and displayed a decreased expression of MK-specific glycoproteins (GPs) and transcription factors. In addition, TGFpi was identified to be involved in the promotion of myelofibrosis and, surprisingly, osteosclerosis in female mice (Figure 3C-3E). It was found that the accumulation of profibrotic MKs in the BM of Mpig6b-mutant mice promoted osteoblast proliferation and differentiation in an estrogen-dependent manner, which could be inhibited by neutralizing TGFpi in vitro.

[0200] Amelioration of both myelofibrosis and osteosclerosis was achieved by conditional deletion of RhoA from G6b-B-deficient MKs and platelets (RhoA- / - / Mpig6bmut,Figure 4A). RhoA mediates the release of secondary mediators during platelet aggregation, but its role in the release of cytokines from MKs has not been investigated, although increased RhoA activity has been linked to reduced TGFJ31 secretion from murine fibroblasts.32The normalization of TGFJ31 levels detected in BM plasma of RhoA- / - / Mpig6bmut mice (Figure 4B), however, indicates that MK secretion is dependent on RhoA-mediated signaling.

[0201] The finding of ameliorated myelofibrosis in RhoA- / - / Mpig6bmut mice in conjunction with the observation of a RhoA / autophagy-dependent release of TGFJ31 from fibroblasts strongly suggests that MKs use a similar mechanism to release cytokines into the BM. The inventors identified that mice with a Pf4-Cre-mediated deletion of the small GTPase Arf6 from MKs exhibit myelofibrosis at 12 months of age(Figure 5A). In contrast, 1-year-old mice lacking Vamp3 displayed unaltered BM morphology, thus underlining that SNARE-dependent vesicular secretion is not the primary pathway of cytokine release from MKs. Both Vamp3 and Arf6 were previously implied in fibrinogen uptake and integrin trafficking,15 51but only Arf6 was implied to participate in autophagosome formation.50

[0202] For a long time, the inaccessibility of the BM hematopoietic compartment hindered the investigation of how platelets are generated in vivo, which was only rendered possible upon the discovery of TPO to induce in vitro maturation of MKs from HSCs.1Using immunofluorescencebased microscopy techniques, the extensive cytoskeletal rearrangements that are necessary to enable initial pseudopod formation and subsequent proplatelet shaft elongation were delineated.56Dyneindependent microtubule sliding rather than directed microtubule assembly was identified to mainly contribute to the lengthening of proplatelets thus unraveling the main mechanisms required for the formation of platelets in vivo.11Moreover, myristoylated alanine-rich C-kinase substrate (MARCKS) to be of importance for the induction of proplatelet formation in mice in vitro and in vivo.57MKs directly interact with vessel-lining endothelial cells and can penetrate the cells to release platelets into the sinusoids (Figure 6). By taking advantage of several inhibitors of actin polymerization such as Latrunculin A, it is demonstrated that an interference with actin filament dynamics indeed blocks proplatelet formation in vitro, thus highlighting the importance of both the actin and the microtubule cytoskeleton in the generation of platelets. These pharmacological studies suggest the actin cytoskeleton is highly relevant for proplatelet formation independent of the BM microenvironment. Moreover, it was recently reinforced that the presence of several distinct subtypes of a-granules that are specifically released upon stimulation of platelets with different agonists.59,60

[0203] Thrombocytopenia is a hallmark of a variety of diseases. For acute needs, platelet transfusions are the only treatment option, however, they are exacerbated due to the short platelet unitshelf-life (around 4-5 days) caused by bacterial contamination or platelet deterioration. To this end, recent research has focused on generating in vitro platelets in order to overcome clinical platelet shortages. A platelet bioreactor microfluidic chip has been established that reproduces proplatelet formation in vitro.61The infusion of mature MKs into the device induces the release of platelet-like cells into a microfluidic channel, from which platelets can then be collected. In addition to enabling visualization of proplatelet formation in an in-vivo-like setting, the biochip provides a scaffold for endothelialization, which allows for the investigation of MK-endothelial cell interactions.

[0204] The formation of extracellular vesicles (EVs) from mature MKs has been characterized.62Although their generation is distinct from platelet EVs, the factors triggering the release of these particles, whether they are distinct from MK granules and their function within the BM microenvironment still remains unknown. Several molecules important for autophagosome formation such as ESCRTs were also described to be essential for EV formation in other cell types.63

[0205] Objectives. An aim of the work described herein is to elucidate the role of MKs in the BM compartment independently of their functions as platelet precursors. The project focuses on these questions: a. What are the underlying mechanisms leading to secretion from MKs in normal physiology? b. Which cell biological pathways are important for cytokine secretion by MKs? c. Can pathophysiological secretion from MKs be inhibited for therapeutic purposes?These questions aim to identify secretion mechanisms in MKs that can be targeted therapeutically to prevent aberrant cytokine secretion. This project identifies novel regulators of BM homeostasis to gain a deeper understanding of the complex signaling mechanisms underlying myelofibrosis progression.

[0206] Is secretory autophagy a cytokine secretion mechanism in MKs?

[0207] Rationale: Analyses of myelofibrosis clusters in patient biopsies and respective mouse models in situ revealed an accumulation of immature MKs around fibrotic areas, which prompted the idea of aberrant cytokine release from immature MKs to account for tissue fibrosis, further supported by the abundant expression of profibrotic cytokines such as TGFpi.64 Lysyl oxidase (LOX) expression was found to be markedly increased in both immature WT MKs as well as MKs derived from myelofibrotic mice (Gatallow ), inhibition of which ameliorates myelofibrosis in vivo.65Although it is known that MKs are major contributors to cytokines within the bone marrow, insights into whether MK cytokines are actively released or diffuse into their extracellular environment in a passive process was still a matter of debate. As such, it was unclear whether the secretion of these cytokines can be targeted therapeutically. In addition, the molecular drivers underlying the possible release of single molecules or specific granule subsets have not been previously identified.

[0208] Hypothesis: Preliminary findings suggest a role for autophagosome formation in the release of TGFJ31 from MKs. The inventors hypothesized that inhibition of autophagosome maturation or ytoskeletal signaling through RhoA affects cytokine release from MKs.

[0209] Experimental approach: First, in vitro-cultured MKs at different maturation stages will be used to analyze changes in expression levels of molecules involved in autophagy regulation by both immunoblotting and quantitative PCR (qPCR) to identify whether secretory autophagy is temporally egulated, comparable to LOX.

[0210] Next, immunofluorescent stainings and confocal microscopy will be used to assess whether colocalization of early / late endosome markers as well as early / late autophagosome markers can be detected and quantified with Arf6, dynamin 2 and TGF[31. Moreover, staining for different granule subsets (von Willebrand factor (vWF) and endostatin for a-granules; mepacrine for dense granules) will visualize whether TGFJ31 in MKs shows a different localization pattern compared to what has been proposed for platelets.66Colocalization will be verified using immunogold labeling on native BM MKs as well as human MKs derived from CD34+ BM progenitor cells. Moreover, using assays similar to those performed on ILip secretion from BM macrophages,47in vitro-matured murine BM MKs will be serum-starved and / or treated with cytoskeletal inhibitors (fasudil, rhosin) (Figure 2) to assess the amount of released cytokines. Cytokine secretion will be analyzed using a murine proteome profiler cytokine array (R&D Systems) or specific enzyme-linked immunosorbent assays for TGFpi and Pf4 (R&D Systems). By using a broad range cytokine array, additional cytokines that rely on unconventional secretion mechanisms and are released in an autophagydependent manner can be identified, since in addition to TGFpi, several other cytokines can contribute to BM homeostasis3,7and are upregulated in myelofibrosis.67To confirm the importance of the identified molecules for MK secretion, proteome profiling will be performed using a cytokine array on BM plasma derived from TPO-deficient mice, which have a significantly decreased amount of mature MKs in the BM and should thus display a reduction in cytokine secretion.

[0211] Coculture of MKs with several BM-derived cells induces their proliferation and / or differentiation.22,25,28The effect of BM plasma derived from myelofibrotic mice (Mpig6bmut ) on the preosteoblastic cell line MC3T3-E1 was determined by analyzing their proliferation using bromodeoxyuridine (BrdU) and differentiation using a qPCR approach. Here, a similar approach will be used to quantitate osteoblast cell differentiation potential upon coculture with the megakaryoblast cell lines MEG01 and SET2 treated with TPO, since MKs were previously shown to enhance and stimulate osteoblast proliferation.25,28To circumvent the effect of direct cell / cell interactions on cell proliferation, which also have been proposed to affect osteoblast differentiation, transwell plates will be used in these assays. In vitro-matured murine MKs will be similarly cocultured with MC3T3-E1 cells.

[0212] Moreover, MK cell lines or primary MKs will be pretreated with the above-mentioned inhibitors, in order to identify whether differences in osteoblast differentiation can be induced thereby or use MK-conditioned media of different culture conditions (with or without inhibitors) on the preosoteoblast cells. As a positive control, MKs derived from myelofibrotic mouse models like Nbeal2- / - or Arf6fl / fl,Pf4Cre ,68which should increase preosteoblast proliferation will be used.

[0213] A previous study established a coculture system of native murine MKs with endosteal HSCs and found HSC proliferation to be increased in the presence of MKs,21. This will be recapitulated using murine cells that will be analyzed by flow cytometry. A protocol for the isolation of CD34+ progenitors as well as native MKs from human femoral heads will be used to establish coculture assays in vitro. Similarly to MC3T3-E1 cells, MKs will be pretreated with autophagy or cytoskeletal inhibitors (Figure 2) to alter cytokine secretion in vitro.

[0214] Expected outcome and alternative approaches: By using the aforementioned approaches the mechanisms underlying physiological MK secretion, which appears to rely on cytoskeletondependent secretory autophagy, will be identified. It is hypothesized that TGFpi release, in contrast to other cytokines like Pf4, is dependent on autophagic activity, inhibition of which should result in markedly reduced levels of TGFpi in cell culture supernatants. In addition to using human umbilical cord blood-derived CD34+ cells for coculture experiments, CRISPR / Cas9-mediated editing of MKs69 can also be used to analyze the role of specific genes on cytokine secretion. Moreover, the technique offers the possibility of stably expressing fluorescently labeled proteins, which will help visualize autophagy / endocytosis and possibly the release of cytokines in vitro.70

[0215] How does defective secretory autophagy / endosomal recycling affect BM homeostasis?

[0216] Rationale: An enhanced release of proinflammatory cytokines from MKs causes fibrotic alterations in a variety of myeloproliferative diseases. The underlying molecular pathways causing the altered secretome, however, are not well studied yet. It was previously published that deficiency of the large GTPase dynamin 2 from MKs results in impaired endocytosis of the TPO receptor c-Mpl eventually culminating in constitutive TPO signaling, the accumulation of immature MKs in the BM and subsequent myelofibrotic alterations.53However, no similar impairment in c-Mpl endocytosis was observed in mice lacking the small dynamin 2 effector GTPase Arf6. 15 Strikingly, by analyzing BM morphology, the inventors identified myelofibrosis in conditional Arf6-deficient mice (Figure 5a), thus suggesting that impaired c-Mpl endocytosis and subsequent constitutive Jak2 signaling might not solely be responsible for myelofibrosis progression in dynamin 2-deficient mice. On the contrary, the inventors demonstrated that Arf6 colocalizes to LC3B-positive autophagosomes that were also positive for TGFpi (Figure 5b). The discovery that Arf6 is mediating membrane transfer during autophagosome formation offers a rationale that might explain the fibrotic alterations observed in mice lacking Arf6.

[0217] Hypothesis: It is hypothesized that a dynamin 2-Arf6 axis regulates physiological MK secretion, which is dysregulated upon lack of one of the molecules, resulting in aberrant cytokine release.

[0218] Experimental approach: It will be characterized how lack of Arf6 from MKs and platelets results in a progressive myelofibrosis. The time course of disease progression will be identified by analyzing bone marrow and spleen morphology in Arf6-deficient and control mice at 6, 9 and 12 months of age by immunofluorescence microscopy (collagen I, IV staining) and immunohistochemistry (reticulin, hematoxylin / eosin (HE)), since 12-month-old mice exhibit fibrotic alterations in the BM.

[0219] Cytokine release into the BM will be analyzed using a proteome profiler cytokine array (R&D Systems), which will help to identify target cytokines besides TGFJ31 that might be involved in promoting fibrosis. Furthermore, a flow cytometry panel will be utilized to investigate MK maturation ex vivo by analyzing glycoprotein expression and polyploidization of native MKs derived from WT and Arf6-deficient mice. To confirm alterations in HSC proliferation between young / aged WT and Arf6-deficient mice, progenitor populations will be analyzed using high dimensional flow cytometry. 71

[0220] Expected outcome and alternative approaches: By analyzing Arf6-deficient mice, it will be identified how lack of the small GTPase from MKs acts as a driver of meyolofibrosis in mice. Since myelofibrosis progression in mice and humans are age -dependent,72it is hypothesized that TGFpi secretion and accumulation are elevated in older mice. To verify the role of Atg proteins in MK secretion, mice conditionally lacking the El enzyme Atg7 (Atg7fl / fl,Pf4Cre ), important for autophagy initiation, will be used to similarly analyze BM morphology, HSC and progenitor distribution and MK maturation. Mice specifically lacking the protein in the hematopoietic compartment display impaired megakaryopoiesis, however, although conditional Pf4-Cre-mediated deficiency does not affect MK maturation, the distribution of HSCs and progenitors was not analyzed in more detail, which will be an alternative approach of the project.

[0221] Can pathophysiological MK secretion he altered using cytoskeletal / autophagy inhibitors?

[0222] Rationale: The treatment options for patients suffering from symptomatic and high-risk PMF are still limited. Most promising results are currently achieved with a monoclonal antibody targeting JAK2, ruxolitinib, that inhibits overactive kinase activity,73however, it has so far only shown intermediate disease-modifying properties. In the longterm, only allogeneic stem cell transplantation permits remission or cure of PMF, but is associated with high incidence morbidity and mortality.74

[0223] Ameliorated myelofibrosis in mice lacking G6b-B and the Rho GTPase RhoA suggests the actin cytoskeleton is essential for the release of granules from profibrotic MKs (Figure 4). In addition, the myelofibrosis identified in Arf6-deficient mice suggests a role for secretory autophagy inMK secretion. As such inhibitors of autophagy and / or the cytoskeleton can potentially be used to target aberrant MK secretion in myelofibrosis.

[0224] Hypothesis: It is hypothesized herein that treatment of myelofibrotic mice with autophagy or cytoskeletal inhibitors will ameliorate disease progression by interfering with cytokine secretion from MKs.

[0225] Experimental approach: A chimeric mouse model, in which lethally irradiated mice are transplanted with colony-forming units that were transduced with the MPZenTPO virus will be used.81TPO overexpression in these mice induces a fast-progressing myelofibrosis that develops within 8 weeks after transplantation of transgenic BM cells. Due to this rapid disease progression, the treatment period of mice with the respective inhibitors will be limited. Mice will only be treated with the inhibitors if the in vitro findings suggest reduced cytokine secretion upon treatment (Figure 2). The following treatment plans can be used, e.g., at concentrations and delivery options that have been described for the respective inhibitors in previous publications.1. The antibibiotic tetracycline, an early autophagy inhibitor, can be administered orally or intraperitoneally (i.p.) . If oral administration is unsuccessful, tetra / doxycy cline (2 mg kg- 1) or its derivative minocycline (100 mg kg- 1) can be injected i.p. daily over 6 consecutive weeks.82,832. The PI3K inhibitor 3 -methyladenine can be injected i.p. twice a week at 30 mg kg- 1 over 6 weeks.843. The ROCK inhibitor Fasudil can be given by oral gavage at 10 to 30 mg kg- 1 daily over 6 consecutive weeks.85,864. Rhosin, an inhibitor of RhoA, can be given i.p. at 30 mg kg- 1 daily for 6 consecutive weeks. +

[0226] Mice will be monitored daily for abnormalities in behavior and assessed for general wellbeing. Treatments will start 2 weeks after transplantation and samples will be retrieved 4, 6 and 8 weeks after transplantation. Disease initiation and progression will be investigated using a hematology analyzer (blood counts), immunohistochemistry (HE; reticulin), immunofluorescence stainings on bone cryosections (collagen I) and cytokine arrays or an enzyme-linked immunosorbent assay for TGF[31 (R&D Systems).

[0227] Expected outcome and alternative approaches: It is hypothesized herein that granule release in the BM of diseased mice is cytokine -specific and driven by cytoskeletal rearrangements (Figure 4). The above-described experiments will address whether secretion is a directed, autophagy / endocytosis-dependent process and how aberrant megakaryopoiesis might hijack secretory pathways. Due to in vitro observations in conjunction with previous data on TGF[3132 and IL 1 P secretion47,88it is hypothesized that inhibition of autophagy reduces the release of TGFpi (and possibly other cytokines) into the BM, preferentially without affecting MK maturation. Treatment of mice that develop a fast-progressing myelofibrosis with inhibitors of secretory autophagy should thuspresent with ameliorated disease outcome. Since little is known about MK secretion the findings - irrespective of the outcome - will improve knowledge of how MKs maintain BM homeostasis and might contribute to the identification of novel targets to prevent myelofibrosis progression.

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[0229] Described herein is the identification of novel pathways involved in the pathogenesis of myeloproliferative neoplasms. Myeloproliferative neoplasms, are rare bone marrow diseases caused by mutations in the thrombopoietin (TPO) receptor MPL or the downstream effector kinase Janus Kinase 2 (JAK2). While it is known that MKs are drivers of disease progression, what causes the aberrant secretion of cytokines from diseased MKs remained unexplored. MKs are large cells primarily residing in the bone marrow that derive from hematopoietic stem cells in a complex differentiation and maturation process. While it was previously assumed that the main function of MKs is the production of platelets, recent studies using single cell RNA Sequencing (scRNA Seq) have revealed different subsets of MKs that additionally serve immune functions and maintain stem cell quiescence. 2-4

[0230] Described herein are three aims: (1) Identify whether secretory autophagy is responsible for cytokine secretion from megakaryocytes, (2) investigate how loss of endosomal recycling via Arf6 affected secretory autophagy in vivo and (3) test whether inhibition of secretory autophagy can ameliorate myelofibrosis (MF) in a preclinical mouse model. The results of each aim is discussed herein.

[0231] Aim 1: Is secretory autophagy a cytokine secretion mechanism in MKs?

[0232] Aim 1 was based on the hypothesis that a distinct secretion pathway of the profibrotic cytokine transforming growth factor [31 (TGFJ31) from fibroblasts. Due to the specific increase in TGF[31 observed in the bone marrow of patients and mouse models with MF, it was further hypothesized that TGFJ31 secretion was independent of classic granule secretion in MKs and could therefore be targeted specifically. In addition to fibroblasts and stromal cells, MKs are main contributors to TGFJ31 levels in the bone marrow niche. To validate this, two thrombocytopenic mouse models were utilized - mice either lacking thrombopoietin (Tpo- / - ) or its receptor MPL (Mpl- / - ). Both strains present with highly reduced MK numbers. When analyzing the bone marrow supernatant for TGFJ31, significantly lower levels of the cytokine were found in both Tpo- / - and Mpl- / - mice, indicating that MKs under steady state markedly contribute to maintaining TGFJ31 levels. In situ, TGF[31 staining negatively correlated with MK size as well as the late MK marker CD42b, suggesting that MKs with a high expression of TGFJ31 are smaller and less mature. This indicates heterogeneity within the MK lineage2,3with one low-ploidy subset of MKs mainly being responsible for the maintenance of hematopoietic stem cells (HSCs) through the secretion of cytokines.

[0233] Next, it was investigated whether TGFJ31 localized to MK a-granules, as previously suggested.5Murine bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) were matured into MKs using TPO. Mature MKs were enriched and stained for a variety of a-granule proteins (platelet factor 4 (Pf4), von Willebrand Factor (vWF)) as well as the TGFJ31 -binding proteinlatency-associated peptide (LAP) or TGFpi directly. In contrast to previous reports, no colocalization of LAP / TGFpi was observed with either of the tested a-granule proteins. Similar results were obtained in platelets. However, LAP did co-localize with the autophagosome membrane marker autophagy marker light chain 3B (LC3B) in native MKs, strongly indicating that TGFpi secretion was indeed autophagy-dependent. Granules were visualized by super-resolution microscopy.

[0234] It was hypothesized that similarly to the cytokine interleukin ip (IL- 1 P) in macrophages and neutrophils, TGFpi secretion might be induced by serum starvation. To test this idea, mature MKs were cultured in serum free media for 3h and TGFp 1 levels assessed in the supernatant and intracellularly. No increased release of TGFpi into the supernatant was observed.

[0235] It was hypothesized that RhoA / ROCKl activity controls secretory autophagy and TGFpi secretion in MKs, a process that could be targetable to prevent fibrosis progression in vivo. To test this in vitro, it was first assessed whether inhibition of RhoA or R0CK1 would affect TGFpi release from MKs. Similarly to the tested autophagy inhibitors, HSPCs were treated with the RhoA inhibitor CCG1423 and the ROCK inhibitor Y27632 on day 0 and MK maturation and TGFpi content analyzed in the cell culture supernatant on day 4. A difference in the number or maturity of MKs was not found (on the contrary, ROCK1 inhibition appeared to mildly enhance MK differentiation), but TGFpi levels in the supernatant were significantly lower than in the DMSO control. This correlated with an increased intracellular retention of TGFpi upon RhoA / ROCKl inhibition as assessed by immunofluorescence. These findings indicate similar mechanisms to account for TGFpi retention upon inhibition of either RhoA and secretory autophagy and indicate therapeutic potential.

[0236] Differences in TGFpi secretion upon treatment of MKs with autophagy inhibitors were observed. The levels were below those observed for Pf4.

[0237] Aim 2: How does defective secretory autophagy / endosomal recycling affect BM homeostasis?

[0238] Different mouse models with defective vesicular trafficking were assessed for differences in TGFpi secretion. Bones were isolated from a variety of mouse models with defective a-granule biogenesis (Nbeal2- / - ), impaired exocytosis (Vamp7 / 8- / - ) and altered endocytosis (Arf6fl / £l pf4Cre).

[0239] While Vamp7 / 8-deficient mice presented with normal bone marrow morphology, initial analyses of the bone marrow of aged Arf6-deficient mice revealed degenerative changes, including collagen accumulations and increased MK numbers. Moreover, Arf6fl / fl pf4Cremice exhibit increased levels of TGFpi in their bone marrow fluid, a finding that was in line with Nbeal2-deficient mice, which develop progressive bone marrow fibrosis.8To properly assess the changes occurring upon lack of Arf6, freshly isolated femurs from aged mice were used for bone marrow profiling by flow cytometry and cryosectioning. An expansion of the HSPC compartment was observed in Arf6fPfl pf4Cremice, accompanied by an increase in MK numbers assessed in femoral cryosections and immunofluorescent stainings.

[0240] To further delineate disease progression, lethally irradiated mice were transplanted with whole bone marrow derived from Arf6- deficient mice and littermate controls. HSPCs, MKs and fibrosis were assessed in mice 6 months after transplantation and an expansion of long-term and shortterm HSCs and an increase in collagen was observed within the bone marrow as well as higher TGF[31 levels in the bone marrow fluid.

[0241] In addition, to these in vivo findings, the effect of Arf6 inhibition on MKs was assessed in vitro using a small molecule inhibitor of Arf6 (NAV 2729). In line with the in vivo findings, NAV 2729 treatment increased MK differentiation, while simultaneously inhibiting their maturation, i.e. MKs treated with NAV 2729 were smaller in size but more abundant. Moreover, intracellular staining for TGFpi revealed reduced TGFpi content, correlating with an enhanced release. These in vitro observations are therefore corroborating the in vivo data, indicating that Arf6 is important for the retention of intracellular TGFp 1.

[0242] Bone marrow derived from mice with an HSC-specific deletion of the autophagyregulating gene, Atg5 (Atg5fl / fl,MxlCre ) were analyzed. Atg5 -deficient mice had mostly unaltered MK morphology and numbers in the bone marrow, however, maturation in vitro was mildly reduced. Moreover, Atg5 -deficient MKs presented with highly reduced intracellular TGFpi levels, correlating with their impairment in autophagosome formation. These initial findings indicated a role of autophagy in TGFpi secretion, which was further tested in Aim 3.

[0243] Aim 3: Can pathophysiological MK secretion he altered using cytoskeletal / autophagy inhibitors?

[0244] To test whether secretory autophagy regulated TGFpi release in vivo, a disease model in which TGFpi secretion is highly dysregulated, mostly due to aberrant MK maturation was used. Myeloproliferative neoplasms in humans are mostly caused by mutations in genes encoding for the TPO receptor MPL, its downstream effector JAK2 or the calcium binding protein Calreticulin. These mutations can cause different diseases: aberrant megakaryopoiesis and platelet production (Essential thrombocythemia, ET), dysfunctional erythropoiesis (Polycythemia vera, ET) or primary MF, leading to collagen and reticulin accumulations in bone marrow and spleen and eventually causing bone marrow failure. Of note, both ET and PV can transform into secondary MF, which dramatically reduces life expectancy for patients.

[0245] A variety of mouse models mimicking MF progression in vivo exist. We utilized a murine stem cell virus (MSCV) construct leading to the expression of mutated MPL (MPLW515L ) in transduced cells, which induces a fast-progressing myeloproliferative disease with thrombocytosis and MF.13Upon lethal irradiation of recipient mice and transplantation of transduced cells, increased TGFpi levels in the bone marrow and within MKs, severe MF and splenomegaly were observed. Analysis of platelet lysates further revealed highly increased basal autophagy (as assessed by LC3Blevels) in MPLW515L - transplanted mice, suggesting enhanced autophagy in the MK and platelet lineage upon neoplastic transformation. scRNA sequencing on MKs from these mice found a similar increase in Mapllc3b expression (the gene encoding for the autophagy marker LC3B) in mutant MKs suggesting enhanced autophagy occurs in both mouse models of MF.

[0246] To test the hypothesis that secretory autophagy regulates TGFJ31 secretion from MKs in vivo, Atg5 -deficient cells were utilized. Both wildtype (WT) and Atg5fl / fl’MxlCreHSPCs were transduced with either an empty eGFP-control vector or the MSCV-IRES-EGFP-MPLW515L vector. Recipient C57BL / 6N mice were lethally irradiated, transplanted and disease progression was allowed for 4 weeks. Complete blood counts were assessed for all groups and while no differences were observed between mice transplanted with WT or Atg5fl / fl MxlCrecells transduced with the control vector, platelet, red blood and white blood cell counts were significantly lower in the Atg5fl / fl MxlCregroup in the MPL model, indicating ameliorated disease. This was verified by flow cytometry, where mice transplanted with Atg5fl / fl.MxK're bone marrow exhibited a lower percentage of eGFP+ CD45+ cells in circulation. In line with the mitigated blood cell counts, reduced spleen size, decreased collagen deposition in bone marrow and spleen, as well as lower TGFJ31 levels in the bone marrow, were observed, highlighting that autophagosome formation appears to be essential for disease progression even beyond cytokine secretion.

[0247] Next, it was examined whether interfering with RhoA / ROCKl inhibition similarly affected disease progression. To use a genetic model, floxed RhoA mice were used. MK- and platelet- selective RhoA-deficient mice were generated, by crossing the floxed RhoA mice with Pf4-Cre mice (commercially available), which express the Cre recombinase under the Pf4 promoter. As previously described for a different Pf4-Cre-mediated RhoA knockout line,15macrothrombocytopenia was observed, thus verifying successful gene excision. HSPCs derived from Cre-negative and -positive mice were used and transduced with MSCV-IRES-EGFP-MPLW515L then transplanted recipient mice, in order to assess fibrosis grade after 4 weeks.

[0248] To surpass the use of a genetic model, the effect of inhibition of the RhoA / ROCKl pathway on disease progression was assessed. It is contemplated herein that ROCK1 activity is necessary for inhibition of TGFpi secretion via autophagy. Thus, the general ROCK inhibitor Y27632 was used treated mice treated for 2 consecutive weeks daily with a dose of 20 mg / kg. A significantly lower platelet and white blood cell counts in Y27632-treated animals, indicative of ameliorated disease, despite an unaltered percentage of eGFP-positive blood cells in circulation. Moreover, levels of TGFp 1 as well as a second cytokine known to be secreted via secretory autophagy, IL- 1 P, were reduced in the bone marrow of Y27632-treated mice. In summary, these findings indicate that aberrant cytokine secretion from MKs in MF can be targeted by inhibiting RhoA / ROCKl -induced secretory autophagy.

[0249] The current gold standard for MF treatment is the JAK2 inhibitor ruxolitinib. Although it ameliorates splenomegaly, fibrosis symptoms are often unresolved, and patients are in danger of developing thrombocytopenia leading to termination of treatment. Moreover, some patients are unresponsive to JAK2 inhibition, thus demanding for novel treatment options. To investigate whether a combination of JAK2 and secretory autophagy inhibition would further ameliorate disease symptoms, mice were treated with either ruxolitinib or both ruxolitinib and Y27632 for 2 weeks daily and blood counts, fibrosis grade and TGFpi levels in the bone marrow were assessed. A significant improvement in spleen size was observed after ruxolitinib treatment, and the effect was even more pronounced after combination treatment with ruxolitinib and Y27632. This was accompanied by significantly lower white blood cell counts in mice treated with the combination therapy as well as reduced collagen deposition in the bone marrow. Only the combination treatment reduced TGFpi levels in the bone marrow compared to both the vehicle and the ruxolitinib group. This indicates the disease-improving features of autophagy / ROCK inhibition in a mouse model of MF.

[0250] It is contemplated herein that human bone marrow organoids16engrafted with human cord blood-derived CD34+ cells transduced with a novel MSCV-MPLW515L construct can be used to assess efficacy and dosing of the treatments described herein.

[0251] Summary

[0252] For a long time, the function of bone marrow-resident megakaryocytes (MKs) was thought to be restricted to platelet production. Recent advances in multi -omics, however, have revealed a wide range of alternative roles of MKs including immunity and maintenance of bone marrow homeostasis. Dysregulation of MKs is a common feature of myeloproliferative neoplasms (MPNs), rare diseases caused by mutations within the thrombopoietin receptor MPL or its downstream effector kinase JAK2. Immature MKs accumulate within the bone marrow and spleen, where they induce fibrotic alterations ultimately leading to pancytopenia and bone marrow failure. The underlying mechanisms leading to enhanced cytokine release from these neoplastic MKs, however, remained elusive and was therefore the main aim of this work.

[0253] TGF[31 co-localized with the autophagy marker light chain (LC) 3B in MKs, while colocalization of TGFpi with a-granule proteins was not observed by super-resolution confocal microscopy. When interfering with secretory autophagy by directly targeting autophagosome formation or by inhibiting the GTPase RhoA or its downstream effector Rho kinase 1 (ROCK1), which were previously shown to promote autophagy, markedly reduced TGFpi secretion in vitro and its concomitant intracellular accumulation were observed.

[0254] Next, it was examined whether secretory autophagy in MKs contributed to myelofibrosis pathogenesis in vivo. To this end, an MPLW515L -driven transplant model was established. Briefly, hematopoietic stem and progenitor cells were transduced with a viral construct carrying a mutation within the thrombopoietin receptor MPL (MSCV-IRES-EGFP-MPLW515L ). Transplantation ofthese cells into lethally irradiated mice leads to rapid development of a myeloproliferative disease. Previous studies revealed that autophagy inhibition can enhance sensitivity of MKs to apoptosis in myelofibrosis suggesting that autophagy in general is important for disease progression. To test this, a mouse model with a conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system (Mxl-Cre+ ; Atg5fl / flmice) was utilized. Atg5 deficiency prevented leukocytosis, MK clustering and aberrant TGFJ31 and IL 1 P secretion in the MPLW515L - transplant model, indicating that autophagy promoted the initiation of myelofibrosis.

[0255] MPLW515L -transplanted mice treated with the ROCK inhibitor Y27632 alone or in combination with the JAK2 inhibitor Ruxolitinib reduced MK clustering, cytokine release and fibrosis in spleens and femurs of treated mice. It is contemplated herein that a RhoA-deficient mouse can be utilized in the MPLW515L -driven transplant model. It is further contemplated that the efficacy and dosing of treatments described herein can be assessed in a human bone marrow organoid engrafted with MPLW515L -transduced cells.

[0256] In summary, the present data indicate that TGFpi and IL 1 P secretion in MKs is distinct from conventional granule secretion and is regulated through secretory autophagy via RhoA / ROCKl. Targeting these pathways in aberrant MKs thus represents a new potential therapeutic pathway in the treatment of MF.

[0257] REFERENCES1. Becker IC, Nagy Z, Manukjan G, et al. G6b-B regulates an essential step in megakaryocyte maturation. Blood Adv. 2022.2. Li JJ, Liu J, Li YE, et al. Differentiation route determines the functional outputs of adult megakaryopoiesis. Immunity. 2024;57(3):478-494 e476.3. Sun S, Jin C, Si J, et al. Single-Cell Analysis of Ploidy and Transcriptome Reveals Functional and Spatial Divergency in Murine Megakaryopoiesis. Blood. 2021.4. Wang H, He J, Xu C, et al. Decoding Human Megakaryocyte Development. Cell Stem Cell. 2021;28(3):535-549 e538.5. Fava RA, Casey TT, Wilcox J, Pelton RW, Moses HL, Nanney LB. Synthesis of transforming growth factor-beta 1 by megakaryocytes and its localization to megakaryocyte and platelet alphagranules. Blood. 1990;76(10): 1946-1955.6. Wang Q, You T, Fan H, et al. Rapamycin and bafilomycin Al alter autophagy and megakaryopoiesis. Platelets. 2017;28(l):82-89.7. Nuchel J, Ghatak S, Zuk AV, et al. TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators. Autophagy. 2018;14(3):465-486.8. Guerrero JA, Bennett C, van der Weyden L, et al. Gray platelet syndrome: proinflammatory megakaryocytes and alpha-granule loss cause myelofibrosis and confer metastasis resistance in mice. Blood. 2014;124(24):3624-3635.9. Bender M, Giannini S, Grozovsky R, et al. Dynamin 2-dependent endocytosis is required for normal megakaryocyte development in mice. Blood. 2015; 125(6): 1014-1024.10. Moreau K, Ravikumar B, Puri C, Rubinsztein DC. Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5 -bisphosphate and phospholipase D. J Cell Biol. 2012;196(4):483- 496.11. Boshans RL, Szanto S, van Aelst L, D'Souza-Schorey C. ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rael and RhoA. Mol Cell Biol. 2000;20(10):3685- 3694.12. Chua BA, Lennan CJ, Sunshine MJ, et al. Hematopoietic stem cells preferentially traffic misfolded proteins to aggresomes and depend on aggrephagy to maintain protein homeostasis. Cell Stem Cell. 2023;30(4):460-472 e466.13. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006;3(7):e270.14. Courdy C, Platteeuw L, Ducau C, et al. Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2(V617F) myeloproliferative neoplasms. Blood Cancer J. 2023;13(l): 106.15. Pleines I, Hagedorn I, Gupta S, et al. Megakaryocyte -specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood. 2012;l 19(4): 1054-1063.16. Khan AO, Rodriguez-Romera A, Reyat JS, et al. Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discov. 2023;13(2):364-385.17. Psaila B, Wang G, Rodriguez-Meira A, et al. Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets. Mol Cell. 2020;78(3):477-492 e478.18. Gleitz HFE, Dugourd AJF, Leimkuhler NB, et al. Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN. Blood.2020;136(18):2051-2064.

Claims

What is claimed herein is:

1. A method of treating a blood cancer in a subject in need thereof, the method comprising administering to the subject a RhoA / ROCK inhibitor.

2. The method of claim 1, wherein the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; belumosudil;RKI-1447; Y-27632; GSK429286A; Y- 30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.

3. The method of claim 1, wherein the RhoA / ROCK inhibitor is selected from the group consisting of:Netarsudil; Y-30141; BA-210; p-elemene; GSK-576371; H-1152; Y-33075;Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001;GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B;AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677; LX-7101; GSK180736; Y-27632; Y-27632 dihydrochloride; Fasudil; TS-f22;Hydroxyfasudil; Belumosudil; and NRL-1049.

4. The method of any one of the preceding claims, wherein the RhoA / ROCK inhibitor is a ROCK1 inhibitor.

5. The method of any one of the preceding claims, wherein the RhoA / ROCK inhibitor is a ROCK1 / 2 inhibitor.

6. The method of any one of the preceding claims, wherein the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is selected from the group consisting of: chroman 1; BAY-549; TCS-7001; GSK269962A; verosudil; Y 33983; AMA-0076;DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622GSK429286A;RKI-1447; ripasudil; SR3677; GSK180736; and Y-27632.

7. The method of any one of the preceding claims, wherein the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is Y-27632.

8. The method of any one of the preceding claims, wherein the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 300 nM or lower.

9. The method of any one of the preceding claims, wherein the RhoA / ROCK inhibitor has an IC50 for R0CK1 of 250 nM or lower.

10. The method of any one of the preceding claims, wherein the subject has or is diagnosed as having a myeloproliferative neoplasm.

11. The method of any one of the preceding claims, wherein the subject has or is diagnosed as having aberrant megakaryopoiesis, essential thrombocythemia, or polycythemia vera.

12. The method of any one of the preceding claims, wherein the subject has or is diagnosed as having Myelofibrosis (MF).

13. The method of any one of the preceding claims, wherein the subject has or is diagnosed as having primary myelofibrosis or secondary myelofibrosis.

14. The method of any one of the preceding claims, wherein the method further comprises administering to the subject a JAK2 inhibitor.

15. The method of claim 14, wherein the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; filgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868.

16. The method of claim 14, wherein the JAK2 inhibitor is ruxolitinib.

17. The method of any one of the preceding claims, wherein the subject is a mammal.

18. The method of any one of the preceding claims, wherein the subject is human.

19. The method of any one of the preceding claims, whereby the amount and / or rate of myelofibrosis in the subject is reduced.

20. The method of any one of the preceding claims, whereby the amount and / or rate of myelofibrosis in the bone marrow and / or spleen of the subject is reduced.

21. A RhoA / ROCK inhibitor, and optionally a JAK2 inhibitor, for use in the treatment of blood cancer.

22. The inhibitor of claim 21, wherein the RhoA / ROCK inhibitor is selected from the group consisting of: fasudil; ripasudil; netarsudil; belumosudil;RKI-1447; Y-27632; GSK429286A; Y- 30141; AT-13148; BA-210; p-elemene; chroman 1; DJ4; GSK-576371; H-1152; hydroxyfasudil; LX-7101; NRL-1049, RKI-1447; TCS-7001; thiazovivin; verosudil; Y-30141; Y-33075; and Y 33983.

23. The inhibitor of claim 21, wherein the RhoA / ROCK inhibitor is selected from the group consisting of:Netarsudil; Y-30141; BA-210; p-elemene; GSK-576371; H-1152; Y-33075;Azaindole 1; Thiazovivin; AT13148; Chroman 1; BAY-549; TCS-7001;GSK269962A; Verosudil; Y 33983; AMA-0076; DJ4; INS-115644; SB 772077B;AMA-0526; AT-13148; CAY10622; GSK429286A; RKI-1447; Ripasudil; SR3677;LX-7101; GSK180736; Y-27632; Y-27632 dihydrochloride; Fasudil; TS-f22; Hydroxyfasudil; Belumosudil; and NRL-1049.

24. The inhibitor of any one of the preceding claims, wherein the RhoA / ROCK inhibitor is a ROCK1 inhibitor.

25. The inhibitor of any one of the preceding claims, wherein the RhoA / ROCK inhibitor is a ROCK1 / 2 inhibitor.

26. The inhibitor of any one of the preceding claims, wherein the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is selected from the group consisting of: chroman 1; BAY-549; TCS-7001; GSK269962A; verosudil; Y 33983; AMA-0076;DJ4; INS-115644; SB 772077B; AMA-0526; AT-13148; CAY10622GSK429286A;RKI-1447; ripasudil; SR3677; GSK180736; and Y-27632.

27. The inhibitor of any one of the preceding claims, wherein the RhoA / ROCK inhibitor, ROCK1 inhibitor, or ROCK1 / 2 inhibitor is Y-27632.

28. The inhibitor of any one of the preceding claims, wherein the RhoA / ROCK inhibitor has an IC50 for R0CK1 of 300 nM or lower.

29. The inhibitor of any one of the preceding claims, wherein the RhoA / ROCK inhibitor has an IC50 for ROCK1 of 250 nM or lower.

30. The inhibitor of any one of the preceding claims, the blood cancer is a myeloproliferative neoplasm.

31. The inhibitor of any one of the preceding claims, wherein the subject has or is diagnosed as having aberrant megakaryopoiesis, essential thrombocythemia, or polycythemia vera.

32. The inhibitor of any one of the preceding claims, the blood cancer is Myelofibrosis (MF).

33. The inhibitor of any one of the preceding claims, the blood cancer is primary myelofibrosis or secondary myelofibrosis.

34. The inhibitor of any one of the preceding claims, wherein the JAK2 inhibitor is selected from the group consisting of: ruxolitinib; tofacitinib; oclacitinib; baricitinib; peficitinib; upadacitinib; febratinib; delgocitinib; filgotinib; abrocitinib; pacritinib; deucravacitinib; ritlecitinib; momelotinib; cerdulatinib; gandotinib; lestaurtinib; cucurbitacin I; and CHZ868.

35. The inhibitor of any one of the preceding claims, wherein the JAK2 inhibitor is ruxolitinib.

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