Methods of identifying patients likely to benefit from treatment with telomerase inhibitor

Abstract

To provide methods of identifying or selecting a patient most likely to benefit from treatment with a telomerase inhibitor, such as imetelstat.SOLUTION: This disclosure provides methods of identifying or selecting a patient most likely to benefit from treatment with a telomerase inhibitor, such as imetelstat, by testing the patient for: a lack of a mutation in each of JAK2, CALR, and MPL; and / or a high-molecular risk (HMR), based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The patient may suffer from myelofibrosis. The disclosure also provides methods of treating myelofibrosis, which include identifying such patients.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to the filing dates of U.S. Provisional Patent Application No. 62 / 712,841, filed on July 31, 2018, and U.S. Provisional Patent Application No. 62 / 772,849, filed on November 29, 2018, according to 35 U.S.C.§119(e), and the disclosures thereof are incorporated herein by reference.

[0002] Sequence Listing This application is electronically filed in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy is named Sequence_Listing.txt and is 356 KB in size.

[0003] This application relates to a method of identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, by identifying patients having a high molecular risk (HMR) based on the absence of mutations in each of JAK2, CALR, and MPL and / or the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The invention also relates to a method of treating myelofibrosis in a subject (i.e., a patient) in need of treatment with a telomerase inhibitor.

Background Art

[0004] Preface Myelofibrosis (MF) is one of the classical BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by clonal myeloproliferation and deregulated kinase signaling. Cervantes, Blood, 124(17):2635-2642(2014). It is also characterized by cytopenia, constitutional symptoms, and splenomegaly and may transform into acute myeloid leukemia. Kuykendall et al., Annals of Hematology, 97:435-431(2018). MF is a Philadelphia chromosome-negative myeloproliferative neoplasm with a poor prognosis, and ruxolitinib, a JAK1 / JAK2 inhibitor, is the currently approved treatment. Ruxolitinib, a Janus kinase (JAK)-1 and JAK-2 inhibitor, is the first-in-class drug licensed in the United States for the treatment of high-risk and intermediate-risk myelofibrosis (MF). Pardanani, et al.; Blood Cancer J.; 4(12):e268(2014). Several other JAK inhibitors are under development, and some are currently undergoing phase 3 clinical trials. Ibid. Other treatment options for MF include allo-SCT, hydroxyurea, interferon, lenalidomide (Revlimid®), and thalidomide. Currently, there are ongoing clinical trials in MF to evaluate selective JAK inhibitors, histone deacetylase / DNA methyltransferase inhibitors, PI3K inhibitors, hedgehog / mammalian target of rapamycin (mTOR) inhibitors, antifibrotic agents, immunomodulators, monoclonal antibodies, and immune checkpoint inhibitors. Shreenivas, et al., Expert Opin Emerg Drugs, 23(1):37-49(2018).

[0005] Other MPNs include essential thrombocythemia (ET) and polycythemia vera (PV). Cervantes (below). MF can occur de novo (primary MF [PMF]) or after previous ET or PV (post-ET or post-PV MF). Ibid. According to Cervantes, MF is a clonal proliferation of multipotent hematopoietic stem cells, and the abnormal cell population releases several cytokines and growth factors into the bone marrow that cause myelofibrosis and stromal changes and form colonies in extramedullary organs such as the spleen and liver. Ibid. Myelofibrosis is associated with mutations in the Janus kinase (JAK) 2 gene (such as the V617F mutation), the thrombopoietin receptor gene (MPL), and the calreticulin gene (CALR). Ibid. It mainly affects the elderly, and according to Cervantes, "Currently, there is no curative therapy other than allogeneic hematopoietic stem cell transplantation (allo-SCT), which can be applied to a small number of patients." Ibid.

[0006] Indeed, according to Langabeer, "The majority of patients with classical myeloproliferative neoplasms (MPNs) of polycythemia vera, essential thrombocythemia, and primary myelofibrosis have a distinct disease driving mutation within the JAK2, CALR, or MPL gene." Langabeer, JAK-STAT, 5:e1248011 (2016). These mutations are so-called driver mutations. Exemplary driver mutations include mutations in JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9. Ibid.

[0007] According to Spiegel, in myelofibrosis (MF), driver mutations in JAK2, MPL, or CALR affect survival and progression to the blast phase and have the greatest risk conferred by the triple-negative status (i.e., wild-type JAK2, MPL, and CALR). Spiegel et al., Blood Adv., 1(20):1729-1738 (2017). Indeed, the absence of JAK2 / MPL / CALR mutations (i.e., triple-negative) is associated with the least favorable outcomes. See also Pardanani, et al. Blood Cancer J.; 4(12):e268 (2014), and Tefferi et al., Blood, 124(16):2507-13 (2014). Furthermore, mutations in high molecular risk (HMR) genes such as ASXL1, EZH2, IDH1 / 2, and SRSF2 are also associated with poor prognosis. Spiegel et al. The increasing number of prognostically adverse / “high molecular risk” mutations (i.e., ASXL1, EZH2, SRSF2, and / or IDH-1 / 2 genes) resulted in progressively worsening survival outcomes independent of conventional risk factors. Guglielmelli et al., Leukemia, 28(9):1804-10 (2014).

[0008] Driver mutations in JAK2, MPL, or CALR are associated with differences in overall survival (OS), either alone or in combination with subclonal mutations in genes such as ASXL1. Spiegel et al. Triple-negative patients without canonical mutations in JAK2, MPL, or CALR have an increased risk of leukemic transformation and shorter OS. Spiegel observed that in patients with myelofibrosis treated with ruxolitinib or momelotinib (JAK1 / 2 inhibitors), these mutations are associated with a shorter time to treatment failure. Ibid. Similarly, "Comparing the clinical characteristics of JAK2-positive, CALR-positive, MPL-positive, and TN MF patients, patients with CALR mutations had significantly lower hemoglobin (mean 8.6 vs 10.7 g / dL; P 5.001) and white blood cell count (mean 11.0 vs 25 g / dL; P 5.033), which is the trend reported in other MPN cohorts." Patel et al., Blood; 126(6):790-797 (2015). Patel et al. observed that patients with three or more mutations treated with ruxolitinib showed an inverse correlation with spleen response and time to treatment discontinuation. Driver mutations or triple-negative (JAK2, MPL, CALR) status are found in patients with myelofibrosis who discontinue treatment with JAK inhibitors. See, for example, Kuykendall et al.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0010] The present invention provides a method for identifying or selecting patients who are most likely to benefit from treatment with a telomerase inhibitor, such as imetelstat, by testing patients for the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes, and / or the presence of a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2). Patients in need of treatment may be suffering from myelofibrosis. The present invention also provides a method for treating myelofibrosis in patients in need of such treatment, the method comprising the step of identifying such patients.

[0011] One embodiment of the present invention is a method for identifying myelofibrosis patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing the patient for (i) a triple negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) selecting the patient if the patient has (i) a triple negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, wherein the selected patients are most likely to benefit from treatment with a telomerase inhibitor.

[0012] An alternative embodiment of the present invention is a method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing a patient for triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and (b) selecting the patient if the patient has triple-negative status, wherein the selected patient is most likely to benefit from treatment with a telomerase inhibitor. An alternative embodiment of the present invention is a method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing a patient for high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) selecting a patient with high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The present invention further provides a method for treating myelofibrosis in triple-negative and / or HMR patients with a telomerase inhibitor such as imetelstat.

[0013] Another embodiment of the present invention is a method for identifying patients with myelofibrosis who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) obtaining a DNA sample from a patient; (b) testing a DNA sample from such a patient for (i) triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (c) selecting a patient if the patient has (i) triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, wherein the selected patients are most likely to benefit from treatment with a telomerase inhibitor. In certain embodiments of the method, the DNA sample is obtained from bone marrow, peripheral blood, or both.

[0014] The DNA sample can be obtained by first obtaining a bone marrow sample, a peripheral blood sample, or both, and then isolating DNA from the bone marrow sample, the peripheral blood sample, or both. In one embodiment, the step of obtaining a DNA sample from a patient comprises obtaining a bone marrow sample from the patient, isolating cells from the bone marrow sample, and extracting DNA from the isolated cells. In another embodiment, the step of obtaining a DNA sample from a patient comprises obtaining a peripheral blood sample from the patient, isolating cells from the peripheral blood sample (e.g., granulocytes), and extracting DNA from the isolated cells.

[0015] Yet another embodiment of the present invention is a method of identifying patients with myelofibrosis who are most likely to benefit from treatment with a telomerase inhibitor, the method comprising testing the patient for (a) triple-negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes, (b) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, or (c) both, wherein the presence of (a), (b), or (c) indicates a patient who is most likely to benefit from treatment with a telomerase inhibitor.

[0016] In any of these methods, the patient may have myelofibrosis. Myelofibrosis can be primary myelofibrosis, myelofibrosis that develops after polycythemia vera (PV-post MF), or myelofibrosis that develops after essential thrombocythemia (ET-post MF). In certain embodiments, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and the JAK inhibitor therapy has "failed" (i.e., the disease was resistant, or the patient was resistant to the therapy, or the patient initially responded to the treatment but the disease recurred). In other embodiments, the patient has received JAK inhibitor therapy and has discontinued the JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0017] The method may also include administering a telomerase inhibitor once such a patient is identified. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0018] When using imetelstat to treat patients identified by these methods, imetelstat is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, and each cycle comprises administering about 7 - 10 mg / kg of imetelstat intravenously once every 3 weeks, administering about 7 - 10 mg / kg of imetelstat intravenously once a week for 3 weeks, administering about 2.5 - 10 mg / kg of imetelstat intravenously once every 3 weeks, or administering about 0.5 - 9.4 mg / kg of imetelstat intravenously once every 3 weeks. In one embodiment, each dosing cycle comprises administering about 7 - 10 mg / kg of imetelstat intravenously once every 3 weeks. In another embodiment, each dosing cycle comprises administering about 9.4 mg / kg of imetelstat intravenously once every 3 weeks.

[0019] When using imetelstat sodium to treat patients identified by these methods, imetelstat sodium is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, and each cycle comprises administering about 7 - 10 mg / kg of imetelstat sodium intravenously once every 3 weeks, administering about 7 - 10 mg / kg of imetelstat sodium intravenously once a week for 3 weeks, administering about 2.5 - 10 mg / kg of imetelstat sodium intravenously once every 3 weeks, or administering about 0.5 - 9.4 mg / kg of imetelstat sodium intravenously once every 3 weeks. In one embodiment, each dosing cycle comprises administering about 7 - 10 mg / kg of imetelstat sodium intravenously once every 3 weeks. In another embodiment, each dosing cycle comprises administering about 9.4 mg / kg of imetelstat sodium intravenously once every 3 weeks.

[0020] Another embodiment of the present invention is a method of treating a patient having myelofibrosis with a telomerase inhibitor such as imetelstat or imetelstat sodium, (i) screening a patient to determine whether such a patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or whether there is a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; (ii) administering a telomerase inhibitor to the patient if such a patient is in a triple-negative status based on the absence of mutations in any of JAK2, CALR, and MPL, and / or if there is a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. Myelofibrosis can be primary myelofibrosis, myelofibrosis developing after polycythemia vera (post-PV MF), or myelofibrosis developing after essential thrombocythemia (post-ET MF). In certain embodiments, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and has failed JAK inhibitor therapy or has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0021] In certain embodiments of the treatment method, the telomerase inhibitor is imetelstat and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising intravenous administration of about 7 - 10 mg / kg of imetelstat once every 3 weeks, intravenous administration of about 7 - 10 mg / kg of imetelstat once a week for 3 weeks, intravenous administration of about 2.5 - 10 mg / kg of imetelstat once every 3 weeks, or intravenous administration of about 0.5 - 9.4 mg / kg of imetelstat once every 3 weeks. In certain embodiments, each dosing cycle comprises intravenous administration of about 7 - 10 mg / kg of imetelstat once every 3 weeks. In another embodiment, each dosing cycle comprises intravenous administration of about 9.4 mg / kg of imetelstat once every 3 weeks.

[0022] In some embodiments of a method for identifying or selecting patients who are most likely to benefit from treatment with a telomerase inhibitor, the method further comprises determining an average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample derived from the patient. In some embodiments of a method for identifying or selecting patients who are most likely to benefit from treatment with a telomerase inhibitor, the method further comprises selecting a patient identified as having an average relative telomere length in target cells present in a biological sample derived from the patient that is at or below the 50th percentile of a relative telomere length range determined from one or more known criteria. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0023] The present disclosure provides a method of treating a patient having myelofibrosis with a telomerase inhibitor, the method comprising administering a telomerase inhibitor to the patient if such patient is in a triple negative status based on the absence of mutations in each of JAK2, CALR, and MPL. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0024] The present disclosure provides a method of treating a patient having myelofibrosis with a telomerase inhibitor, the method comprising administering a telomerase inhibitor to the patient if such patient is in a triple negative status based on the absence of mutations in each of JAK2, CALR, and MPL and / or has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0025] The present disclosure provides a method of treating a patient having myelofibrosis with a telomerase inhibitor, wherein such patient has the following characteristics: (a) The average relative telomere length of target cells present in a biological sample derived from an individual, determined to be below the 50th percentile of a relative telomere length range determined from one or more known references. (b) A triple-negative status, based on the absence of mutations in each of JAK2, CALR, and MPL. (c) Provided is a method that includes administering a telomerase inhibitor to a patient when the patient has one or more of the high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0026] The present disclosure provides a method for identifying a subject having myelofibrosis (MF) for treatment with a telomerase inhibitor, the method including measuring the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor and comparing the hTERT expression level in the biological sample to the baseline hTERT expression level before administration of the telomerase inhibitor, wherein a decrease in the hTERT expression level in the biological sample identifies patients likely to benefit from treatment with the telomerase inhibitor.

[0027] The present disclosure provides a method for treating myelofibrosis (MF), the method including administering an effective amount of a telomerase inhibitor to a subject in need thereof and evaluating the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0028] The present disclosure provides a method for monitoring therapeutic efficacy in a subject having myelofibrosis (MF), the method comprising measuring the hTERT expression level in a biological sample obtained from the patient after administration of a telomerase inhibitor, and comparing the hTERT expression level in the biological sample with the baseline hTERT expression level before administration of the telomerase inhibitor, wherein a decrease of 50% or more in the hTERT expression level in the biological sample identifies a subject likely to benefit from treatment with the telomerase inhibitor. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0029] The present disclosure provides a method of selecting patients most likely to benefit from treatment with a telomerase inhibitor, the method comprising assaying a patient for mean relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a patient-derived biological sample, and selecting the patient if the patient has a mean relative telomere length in target cells present in a patient-derived biological sample that is determined to be at or below the 50th percentile of a relative telomere length range determined from one or more known criteria, wherein the selected patients are most likely to benefit from treatment with the telomerase inhibitor.

[0030] The present disclosure provides a method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, the method comprising obtaining a biological sample from the patient, determining the mean relative length by analyzing the relative length of telomeric nucleic acids in target cells present in the patient-derived biological sample, and identifying the patient if the patient has a mean relative telomere length in target cells present in a patient-derived biological sample that is determined to be at or below the 50th percentile of a relative telomere length range determined from one or more known criteria, wherein the identified patients are most likely to benefit from treatment with the telomerase inhibitor.

[0031] The present disclosure provides a method of treating a patient having myelofibrosis with a telomerase inhibitor, the method comprising administering the telomerase inhibitor to the patient when target cells present in a biological sample derived from the patient are determined to have an average relative telomere length that is at or below the 50th percentile of a relative telomere length range determined from one or more known criteria. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0032] The present disclosure provides a method of monitoring treatment efficacy in a subject having myelofibrosis (MF), the method comprising measuring the hTERT expression level in a biological sample obtained from the patient after administration of a telomerase inhibitor and comparing the hTERT expression level in the biological sample to a baseline hTERT expression level prior to administration of the telomerase inhibitor, wherein a decrease of 50% or more in the hTERT expression level in the biological sample identifies a subject likely to benefit from treatment with the telomerase inhibitor. In certain embodiments, the hTERT expression level measured or evaluated is the hTERT RNA expression level. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium.

[0033] The present disclosure provides a method of identifying a patient having myelofibrosis (MF) for treatment with a telomerase inhibitor, the method comprising measuring the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor and comparing the hTERT expression level in the biological sample to a baseline hTERT expression level prior to administration of the telomerase inhibitor, wherein a decrease in the hTERT expression level in the biological sample identifies a patient likely to benefit from treatment with the telomerase inhibitor.

[0034] The present disclosure provides a method for monitoring therapeutic efficacy in a subject having myelofibrosis (MF), the method comprising measuring the telomerase activity level in a biological sample obtained from the patient after administration of a telomerase inhibitor and comparing the telomerase activity level in the biological sample with the baseline telomerase activity level before administration of the telomerase inhibitor, wherein a decrease of 50% or more in the telomerase activity level in the biological sample identifies a subject likely to benefit from treatment with the telomerase inhibitor. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, imetelstat is imetelstat sodium. The present invention provides, for example, the following items. (Item 1) A method of selecting a patient most likely to benefit from treatment with a telomerase inhibitor, (a) examining the patient for triple negative status, said triple negative status including no mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes, and / or (b) examining the patient to determine whether the patient has HMR, having HMR including the presence of a mutation in at least one gene selected from the group consisting of additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2), and / or (c) examining the patient for average relative telomere length by analyzing the relative length of the telomeric nucleic acids of the target cells present in a biological sample from the patient, and the patient being if the patient has triple negative status, or if the patient has HMR, or selecting when the average relative telomere length of target cells present in a biological sample derived from the patient is determined to be below the 50th percentile of a relative telomere length range determined from one or more known criteria; The method, wherein the selected patient is most likely to benefit from treatment with a telomerase inhibitor. (Item 2) The method according to item 1, comprising examining a patient for triple negative status and selecting the patient if the patient has no mutations in each of the JAK2, CALR, and MPL genes. (Item 3) The method according to item 1, comprising examining the patient to determine whether the patient has HMR, and selecting the patient including the presence of a mutation in at least one gene selected from ASXL1, EZH2, SRSF2, and IDH1 / 2. (Item 4) The method according to item 1, wherein examining the patient for average relative telomere length comprises examining by analyzing the relative length of telomeric nucleic acids of target cells present in a biological sample derived from the patient, and selecting the patient when the patient has the average relative telomere length of target cells present in a biological sample derived from the patient determined to be below the 50th percentile of a relative telomere length range determined from one or more known criteria. (Item 5) The method according to any one of items 1 to 4, wherein the myelofibrosis is selected from the group consisting of primary myelofibrosis, myelofibrosis developing after polycythemia vera (post-PV MF), and myelofibrosis developing after essential thrombocythemia (post-ET MF). (Item 6) The method according to any one of items 1 to 5, wherein the patient has not previously received JAK inhibitor therapy. (Item 7) The patient has received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. who has received JAK inhibitor therapy and has relapsed, or who has received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance, the method according to any one of items 1 to 5. (Item 8) The method according to any one of items 1 to 7, wherein the telomerase inhibitor is imetelstat. (Item 9) The method according to any one of items 1 to 8, further comprising obtaining a sample containing DNA from the patient, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. (Item 10) Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, (a) the patient is determined to have a triple-negative status, which includes no mutations in each of the JAK2, CALR, and MPL genes, and / or (b) the patient is determined to have high molecular risk (HMR), which includes the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2, and IDH1 / 2, and / or (c) the patient having myelofibrosis, wherein the cells present in a biological sample derived from the patient are determined to have an average relative telomere length that is at or below the 50th percentile of a relative telomere length range determined from one or more known criteria. (Item 11) The use according to item 10, wherein the patient is determined to have a triple-negative status, which includes no mutations in each of the JAK2, CALR, and MPL genes. (Item 12) The use according to item 10, wherein the patient is determined to have high molecular risk (HMR), which includes the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2, and IDH1 / 2. (Item 13) The use according to item 10, wherein the patient has myelofibrosis and the cells present in the biological sample derived from the patient have an average relative telomere length determined to be 50th percentile or less of a relative telomere length range determined from one or more known criteria. (Item 14) The use according to any one of items 10 to 13, wherein the myelofibrosis is selected from the group consisting of primary myelofibrosis, myelofibrosis developing after polycythemia vera (MF after PV), and myelofibrosis developing after essential thrombocythemia (MF after ET). (Item 15) The use according to any one of items 10 to 14, wherein the patient has never received JAK inhibitor therapy before. (Item 16) The patient is who has received JAK inhibitor therapy and was resistant to JAK inhibitor therapy, who has received JAK inhibitor therapy and has relapsed, or who has received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. The use according to any one of items 10 to 14. (Item 17) The method according to any one of items 10 to 16, wherein the telomerase inhibitor is imetelstat.

Brief Description of the Drawings

[0035] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the invention, the figures demonstrate embodiments of the invention. However, it should be understood that the invention is not limited to the exact arrangements, examples, and instruments shown.

[0036]

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DETAILED DESCRIPTION OF THE INVENTION

[0037] This application is based on the discovery that patients who have myelofibrosis and are triple-negative (i.e., have no mutations in each of JAK2, CALR, and MPL) and / or are in the high molecular risk (HMR) category based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2 can benefit from treatment with a telomerase inhibitor such as imetelstat or imetelstat sodium. Patients with mutations in the ASXL1, EZH1, IDH1 / 2, and SRSF2 genes have an increased risk of early death or leukemic transformation. These patients typically do not benefit from treatment using conventional therapies such as JAK inhibitors. Gisslinger et al., Blood, 128:1931 (2016). Thus, the fact that these patients can benefit from treatment with a telomerase inhibitor is unexpected and surprising.

[0038] Accordingly, the present application provides a method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor such as imetelstat. The method involves examining or identifying a patient to determine whether the patient has a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or whether the patient has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The present application also provides a method for treating myelofibrosis with a telomerase inhibitor such as imetelstat, the method comprising identifying a patient who has a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or who has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes ASXL1, EZH2, SRSF2, and IDH1 / 2. Such patients are most likely to benefit from treatment with a telomerase inhibitor. The telomerase inhibitor (e.g., imetelstat) is then administered to the patient. For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate particular features, embodiments, or applications of the invention.

[0039] A. Definitions As used herein, mutations in Additional Sex Combs-Like 1 (ASXL1), Enhancer of Zeste Homolog 2 (EZH2), Serine and Arginine Rich Splicing Factor 2 (SRSF2), and Isocitrate Dehydrogenase 1 / 2 (IDH1 / 2) shall include any mutations in these genes that affect survival and disease progression in patients with myelofibrosis. Further, as used herein, IDH1 / 2 shall include IDH1 and IHD2. Exemplary mutations can be found in the following publications, each of whose disclosures is incorporated herein by reference for its disclosure of gene mutations associated with myelofibrosis. Langabeer, JAK-STAT, 5:e1248011 (2016), Cervantes, Blood; 124(17):2635-2642 (2014), Patel et al., Blood; 126(6):790-797 (2015), Spiegel et al., Blood Adv., 1(20):1729-1738 (2017), Newburry et al., Blood, 130(9):1125-1131 (2017), Kuykendall et al. Annals of Hematology, 97:435-431 (2018). Exemplary sequences are as follows: High Molecular Risk (HMR) can be determined based on the presence of a mutation in at least one of the following genes: for example, the ASXL1 gene having the nucleic acid sequence of SEQ ID NO: 5, for example, the EZH2 gene having the nucleic acid sequence of SEQ ID NO: 6, for example, the SRSF2 gene having the nucleic acid sequence of SEQ ID NO: 7, for example, the IDH1 gene having the nucleic acid sequence of SEQ ID NO: 8, the IDH2 gene having the nucleic acid sequence of SEQ ID NO: 9, and combinations thereof.

[0040] In some embodiments, the target mutations in the ASXL1 gene include mutations at Q575, Q588, Y591, Q592, S604, L614, Q623, A627, E635, T638, A640, G646, G658, R678, C687, D690, R693, Y700, G704, E705, Q708, G710, L721, E727, V751, P763, Q780, W796, V807, T822, K825, S846, D855, C856, L857, L885, L890, S903, S970, Y974, R965, G967, V962, L992, S1028, Q1039, R1073, E1102, H1153, S1209, S1231, A1312, F1305, P1377, R1415, and I1436. In some embodiments, the mutations are Q575X mutation, Q588X, Y591X mutation, Y591N mutation, Q592X mutation, S604F mutation, L614F mutation, Q623X mutation, A627G mutation, E635R mutation, T638V mutation, A640G mutation, G646W mutation, G658X mutation, R678K mutation, C687R mutation, C687V mutation, D690G mutation, R693X mutation, Y700X mutation, G704R mutation, G704W mutation, E705X mutation, Q708X mutation, G710E mutation, L721C mutation, E727X mutation, V751L mutation, P763R mutation, Q780X mutation, W796X mutation, W796G mutation, V807F mutation, T822H mutation, K825X mutation, S846Q mutation, D855A mutation, C856X mutation, L857R mutation, L885X mutation, L890F mutation, S903I mutation, S970N mutation, Y974X mutation, R965X mutation, G967del mutation, V962A mutation, L992Q mutation, S1028R mutation, Q1039L mutation, R1073C mutation, E1102D mutation, H1153R mutation, S1209I mutation, S1231F mutation, A1312V mutation, F1305W mutation, P1377S mutation, R1415Q mutation, and / or I1436M mutation.

[0041] In some embodiments, the mutations of interest in the EZH2 gene include mutations at W60, R63, P312, F145, N182, R288, Q328, Q553, R566, T573, R591, R659, D677, V679, R690, A702, V704, E726, D730 and / or Y733. In some embodiments, the mutations are W60X mutation, R63X mutation, P312S mutation, F145S mutation, N182D mutation, R288Q mutation, Q328X mutation, Q553X mutation, R566H mutation, T573I mutation, R591H mutation, R659K mutation, D677H mutation, V679M mutation, R690H mutation, A702V mutation, V704L mutation, E726V mutation, D730X mutation, and / or Y733X mutation.

[0042] In some embodiments, the mutations of interest in the SRSF2 gene include the mutation at P95. In some embodiments, the mutations are P95H mutation, P95L mutation or P95R mutation.

[0043] In some embodiments, the mutations of interest in the IDH1 / 2 gene include mutations at R132 and / or R140. In some embodiments, the mutations are R132G mutation, R132H mutation or R140Q mutation.

[0044] In certain embodiments, the mutations of interest include the mutations described below:

Table A

[0045] As used herein, "triple negative status", "triple negative", or "TN" shall refer to patients without mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. The triple negative status can be determined based on, for example, no mutations in the JAK2 gene having the nucleic acid sequence of SEQ ID NO: 2, the CALR gene having the nucleic acid sequence of SEQ ID NO: 3, and the MPL gene having the nucleic acid sequence of SEQ ID NO: 4, respectively.

[0046] In certain embodiments, the triple negative status includes no mutations in the JAK2 gene, such as mutations at G335, F556, G571, V617, and / or V625. For example, the triple negative status can include no G335D mutation, F556V mutation, G571S mutation, V617F mutation, and / or V625S mutation in the JAK2 gene.

[0047] In certain embodiments, the triple negative status includes no mutations in the MPL gene, such as mutations at T119, S204, P222, E230, V285, R321, S505, W515, Y591, and / or R592. For example, the triple negative status can include no T119I mutation, S204F mutation, S204P mutation, P222S mutation, E230G mutation, V285E mutation, R321W mutation, S505N mutation, W515R mutation, W515L mutation, Y591N mutation, and / or R592Q mutation in the MPL gene.

[0048] In certain embodiments, the triple negative status includes no mutations in the CALR gene, such as mutations at L367, K368, E381, K385, and / or E396. For example, the triple negative status can include no L367T mutation, K368R mutation, K385N mutation, E381A mutation, and / or E396del mutation in the CALR gene.

[0049] In certain embodiments, exemplary mutations include those described below:

Table B

[0050] As used herein, a patient may have "failed" JAK inhibitor therapy when the disease was resistant, or the patient was resistant to treatment, or when the patient was initially responsive to treatment but the disease recurred.

[0051] As used herein, when referring to measurable values such as amounts, durations of time, etc., the term "about" means that for the purposes of practicing the disclosed methods, it encompasses variations of ±20% to ±0.1%, preferably ±20% to ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value.

[0052] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to patients such as mammals (a salt having a counterion having acceptable mammalian safety for a given dosing regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases, as well as pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, the salt being derived from various organic and inorganic counterions well known in the art, including, by way of example only, sodium, etc., and when the molecule contains a basic functional group, including salts of organic or inorganic acids such as hydrochloride. Pharmaceutically acceptable salts for the purposes include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, cholinate, ethylenediamine, lysine, lithium, magnesium, meglumine, procaine, potassium, sodium, tromethamine, N-methylglucamine, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, zinc, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine salts.

[0053] The term "the salt(s)" means a compound formed when the proton of an acid is replaced by a cation such as a metal cation or an organic cation. Preferably, the salt is a pharmaceutically acceptable salt. By way of example, salts of the present compound include those in which the conjugate base of an inorganic or organic acid is used as the anionic component of the salt, and the compound is protonated by an inorganic or organic acid to form a cation. Examples of the desired salts include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, cesium, cholinate, ethylenediamine, lithium, magnesium, meglumine, procaine, N-methylglucamine, piperazine, potassium, sodium, tromethamine, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine salts. It should be understood that for any of the oligonucleotide structures shown herein that include the backbone of the internucleoside linkage, such oligonucleotides may include any convenient salt form. In some embodiments, for simplicity, the acidic form of the internucleoside linkage is shown. In some cases, the salt of the subject compound is a monovalent cation salt. In certain cases, the salt of the subject compound is a divalent cation salt. In some cases, the salt of the subject compound is a trivalent cation salt. "Solvate" refers to a complex formed by the combination of solvent molecules with the molecules or ions of a solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0054] "Stereoisomers" and "stereoisomer" refer to compounds that have the same atomic connectivity but different atomic arrangements in space. Examples of stereoisomers include, for example, cis-trans isomers, E and Z isomers, enantiomers, and diastereomers. It should be understood that for any of the groups disclosed herein that contain one or more substituents, such groups do not contain any substitution or substitution pattern that is sterically non-practical and / or synthetically unrealizable. All stereoisomers are intended to be included within the scope of the present disclosure.

[0055] One of ordinary skill in the art will understand that other tautomeric arrangements of the groups described herein are possible. It should be understood that all tautomeric forms of the subject compounds are encompassed by the structures in which one possible tautomeric arrangement of the groups of the compound is described, even if not specifically shown.

[0056] It is intended to include solvates of pharmaceutically acceptable salts of tautomers of stereoisomers of the subject compounds. These are intended to be included within the scope of the present disclosure.

[0057] Before a particular embodiment is described in more detail, it should be understood that the present invention is not limited to the particular embodiments described, and thus can of course vary. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0058] When ranges of values are provided, each intervening value, unless the context clearly dictates otherwise, is to be understood as being included within the invention to a tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated value or intervening value within that range. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and also within the invention, subject to any specifically excluded limitations within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included within the invention.

[0059] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, representative, exemplary methods and materials are described herein.

[0060] All publications and patents cited herein are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe methods and / or materials in connection with the cited publications. The citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention has no right to antedate such publication by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates, which may need to be independently verified.

[0061] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this description is intended to serve as antecedent basis for the use of exclusive terms, such as "solely", "only", etc., or the use of "negative" limitations in connection with the recitation of claim elements.

[0062] Each of the individual embodiments described and illustrated herein has separate components and features, which can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of recited events, or any other order that is logically possible.

[0063] B. Identification of Patients Most Likely to Benefit from Treatment with Telomerase Inhibitors In one aspect, the disclosure provides a method of identifying or selecting patients with myelofibrosis who are most likely to benefit from treatment with a telomerase inhibitor. The method relies on identifying patients with high molecular risk (HMR) based on a triple-negative status (patients without mutations in each of the JAK2, CALR, and MPL genes) or the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. These triple-negative patients or HMR patients are most likely to benefit from treatment with a telomerase inhibitor such as imetelstat or imetelstat sodium.

[0064] Myelofibrosis can be primary myelofibrosis, myelofibrosis developing after polycythemia vera (post-PV MF), or myelofibrosis developing after essential thrombocythemia (post-ET MF). In certain embodiments, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and has failed JAK inhibitor therapy (i.e., the disease was resistant or the patient was resistant to the therapy, or initially responded to treatment but the disease recurred). In other embodiments, the patient has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. In yet alternative embodiments, the patient has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy.

[0065] In one embodiment, the patient has received JAK inhibitor therapy and the myelofibrosis was resistant to JAK inhibitor therapy. In another embodiment, the patient has received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. In another embodiment, the patient has received JAK inhibitor therapy and the patient has recurred. In an alternative embodiment, the patient has received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0066] In one embodiment, the present invention provides a method of selecting patients who are most likely to benefit from treatment with a telomerase inhibitor by testing for one or more of the triple negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes (i.e., the absence of any mutations). In that embodiment, patients may also be tested for high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In another embodiment, the present invention is a method of selecting patients who are most likely to benefit from treatment with a telomerase inhibitor, the method comprising testing for triple negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes (i.e., the absence of any mutations), and / or testing for high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In another embodiment, the present invention is a method of identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, the method comprising testing the patient for (a) triple negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes, (b) high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, or (c) both. In this embodiment, the presence of (a), (b), or (c) indicates a patient who is most likely to benefit from treatment with a telomerase inhibitor.

[0067] Another embodiment of the present invention is a method of identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, the method comprising a. testing the patient for the following, i. triple negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. testing for high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, b. where the patient has i. a triple-negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes, and / or ii. selecting the patient if they have a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, The selected patients are the ones most likely to benefit from treatment with a telomerase inhibitor.

[0068] Yet another embodiment of the present invention is a method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient for a triple-negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes, selecting the patient if they have a triple-negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes, wherein the selected patients are the ones most likely to benefit from treatment with a telomerase inhibitor. In one embodiment, the method also comprises testing the patient for high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, and selecting the patient if they have HMR.

[0069] In certain embodiments of any of these methods, triple-negative patients lack mutations in the coding regions (exons) of the JAK2, CALR, and MPL genes.

[0070] Furthermore, in other embodiments of any of these methods, high molecular risk (HMR) is determined by the presence of mutations in the coding regions (exons) of at least one of the ASXL1, EZH2, SRSF2, and IDH1 / 2 genes.

[0071] In certain embodiments, high molecular risk (HMR) is determined by detecting the presence of mutations in ASXL1, EZH2, SRSF2, or IDH1 / 2, or combinations thereof. In some embodiments, the method includes detecting the presence of a mutation in ASXL1. In some embodiments, the method includes detecting the presence of a mutation in EZH2. In some embodiments, the method includes detecting the presence of a mutation in SRSF2. In some embodiments, the method includes detecting the presence of a mutation in IDH1 / 2. In some embodiments, the method includes detecting the presence of mutations in ASXL1 and EZH2. In some embodiments, the method includes detecting the presence of mutations in ASXL1 and SRSF2. In some embodiments, the method includes detecting the presence of mutations in ASXL1 and IDH1 / 2. In some embodiments, the method includes detecting the presence of mutations in EZH2 and SRSF2. In some embodiments, the method includes detecting the presence of mutations in EZH2 and IDH1 / 2. In some embodiments, the method includes detecting the presence of mutations in SRSF2 and IDH1 / 2. In some embodiments, the method includes detecting the presence of mutations in ASXL1, EZH2, and SRSF2. In some embodiments, the method includes detecting the presence of mutations in ASXL1, EZH2, and IDH1 / 2. In some embodiments, the method includes detecting the presence of mutations in EZH2, SRSF2, and IDH1 / 2. In some embodiments, the method includes detecting the presence of mutations in ASXL1, EZH2, SRSF2, and IDH1 / 2. In yet another embodiment of the invention, the invention provides a method of identifying or selecting patients in a patient population who are most likely to benefit from treatment with a telomerase inhibitor. In this method, the patient population is screened for patients having mutations in each of the JAK2, CALR, and MPL genes, and triple-negative patients within the population are identified. In an alternative embodiment, the method relies on the identification of triple-negative patients lacking canonical mutations in each of JAK2, MPL, and CALR.

[0072] In certain embodiments, the method also includes the step of collecting a patient DNA sample. The patient sample can be collected from a DNA sample obtained from bone marrow, peripheral blood, or both. Thus, in certain embodiments, the method of the invention includes obtaining a blood sample from a patient and isolating (extracting) DNA from the patient's blood sample. The method may also include the step of isolating cells (e.g., granulocytes) from the patient's blood sample. Similarly, the method of the invention includes obtaining a bone marrow sample and isolating (extracting) DNA from the bone marrow sample. The method may also include the step of isolating cells from the patient's bone sample.

[0073] The patient DNA sample is examined using conventional techniques for the presence or absence of mutations in each of the JAK2, CALR, and MPL genes. Alternatively, the patient DNA sample is examined using conventional techniques for the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In certain embodiments, the patient DNA sample is examined for (i) the presence or absence of mutations in each of the JAK2, CALR, and MPL genes, and (ii) the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0074] In certain embodiments, the examination of the DNA sample can be a next-generation sequencing assay using the Illumina MiSeq platform, as described in Patel et al., Blood; 126(6):790-797 (2015), the disclosure of which related to the DNA sample examination is incorporated herein by reference.

[0075] C. Pharmacodynamics (PD) The present disclosure is based in part on a pharmacodynamic effect demonstrating the association between the response to telomerase inhibition therapy in a subject having myelofibrosis and the decrease in telomerase hTERT expression levels in the subject from baseline levels. In some cases, in subjects who achieved a clinical response (spleen or symptoms) to telomerase inhibition therapy at week 24, a higher percentage of subjects achieved a decrease in hTERT RNA expression levels of 50% or more than those who did not achieve a response.

[0076] The present disclosure provides for the stratification and selection of patients likely to benefit from telomerase inhibition therapy for myelofibrosis and methods of monitoring response, recurrence, and prognosis in subjects undergoing treatment.

[0077] Aspects of the present disclosure include methods of selecting a subject having myelofibrosis (MF) for treatment with a telomerase inhibitor and methods of treating MF. Also provided are methods of monitoring treatment efficacy in a subject having MF. In some cases, the pharmacodynamic effect upon which embodiments of the methods of the subject are based is a decrease in hTERT RNA expression of 50% or more, such as 60% or more, 70% or more, 80% or more, or 90% or more.

[0078] Telomerase ribonucleoprotein consists of components or subunits, two of which are telomerase RNA template (hTR) and telomerase reverse transcriptase protein (hTERT). The hTERT expression level can be evaluated, determined, and / or measured using any convenient method. Various methods can be applied to amplify, detect, and measure the mRNA of telomerase components or related proteins in body fluids. Methods and assays for the purpose of being adaptable for use in the method of interest include, but are not limited to, real-time quantitative RT-PCR assays based on TaqMan fluorescence method, immunohistochemistry for protein expression, as well as those described in U.S. Patent No. 6,607,898, Bieche et al., Clin. Cancer Res February 1 2000(6)(2)452-459, Terrin et al. (「Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and delineates subgroups of patients with the same igVH mutation status and different outcome.」Leukemia 2007;21:965-972), and Palma et al. (「Telomere length and expression of human telomerase reverse transcriptase splice variants in chronic lymphocytic leukemia.」Experimental Hematology 2013;41:615-626).

[0079] The hTERT expression level can be evaluated or measured in any convenient target cell or biological sample. The target cell can be any convenient cell of the patient, including but not limited to cells of the patient's bone marrow or peripheral blood. In some cases, the target cell is isolated from the patient's bone marrow sample. In some cases, the target cell is isolated from the patient's peripheral blood sample. The target cell can be a granulocyte.

[0080] The hTERT RNA expression level can be evaluated or measured in an RNA sample using any convenient method. The RNA sample can be obtained by first obtaining a bone marrow sample, a peripheral blood sample, or both, and then isolating RNA from the bone marrow sample, the peripheral blood sample, or both. In one embodiment, the step of obtaining a sample from a patient includes obtaining a bone marrow sample from the patient, isolating cells from the bone marrow sample, and extracting RNA and / or DNA from the isolated cells. In another embodiment, the step of obtaining an RNA sample from a patient includes obtaining a peripheral blood sample from the patient, isolating cells from the peripheral blood sample (e.g., granulocytes), and extracting RNA and / or DNA from the isolated cells.

[0081] D. Treatment Aspects of the disclosure are based on the absence of any mutations in the JAK2, CALR, and MPL genes (i.e., these genes without mutations or lacking mutations), having a triple-negative status, and / or the following genes: A method of treating myelofibrosis in a subject (i.e., a patient) in need thereof, having a high molecular risk (HMR) based on the presence of a mutation in at least one of ASXL1, EZH2, SRSF2, and IDH1 / 2 is included. One embodiment of the present invention is a method of treating myelofibrosis in a subject (i.e., a patient) in need thereof, having a triple-negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes (i.e., no mutations or lacking mutations in these genes). In one embodiment, the myelofibrosis is primary myelofibrosis. In another embodiment, the myelofibrosis is myelofibrosis developing after polycythemia vera (PV-post MF). In an alternative embodiment, the myelofibrosis is myelofibrosis developing after essential thrombocythemia (ET-post MF).

[0082] In certain embodiments of the treatment method, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and the JAK inhibitor therapy has "failed" (i.e., the disease was resistant or the patient was resistant to the therapy, or initially responded to the treatment but the disease recurred). In an alternative embodiment of the treatment method, the patient has previously received JAK inhibitor therapy and has discontinued the JAK inhibitor therapy due to treatment-related toxicity or intolerance. In certain embodiments, the treatment method further comprises premedication with diphenhydramine (25 - 50 mg) and hydrocortisone (100 - 200 mg), or their equivalents.

[0083] The subject is a mammal in need of treatment for cancer. Generally, the subject is a human patient. In some embodiments of the present invention, the subject can be a non-human mammal such as a non-human primate, an animal model (e.g., an animal such as a rat used for screening, characterizing, and evaluating drugs), and other mammals. As used herein, the terms "patient", "subject", and "individual" are used interchangeably.

[0084] As used herein, and as is well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, reduction or improvement of one or more symptoms, decrease in the degree of disease, stabilization of the disease (i.e., not worsening), prevention of the spread of the disease, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean extending the survival period as compared to the expected survival period in the absence of treatment.

[0085] E. Telomerase inhibitors Using the methods of the present invention, it is possible to identify patients who are most likely to benefit from treatment with any convenient telomerase inhibitor. Furthermore, any convenient telomerase inhibitor can be found for use in the treatment of a subject. In some embodiments, the telomerase inhibitor is an oligonucleotide having telomerase inhibitory activity, particularly an oligonucleotide defined in WO2005 / 023994 and / or WO2014 / 088785, the disclosures of which are hereby incorporated by reference in their entirety. In some cases, one or more telomerase inhibitors (e.g., two or three telomerase inhibitors) can be administered to a mammal to treat a hematological malignancy.

[0086] Imetelstat In certain embodiments, the telomerase inhibitor is imetelstat, including its tautomers and its salts, such as pharmaceutically acceptable salts. Imetelstat is a novel first-in-class telomerase inhibitor with clinical activity in hematological malignancies (Baerlocher et al., NEJM 2015;373:920-928, Tefferi et al., NEJM 2015;373:908-919) (shown below):

Chemical formula

[0087] In certain cases, the telomerase inhibitor is imetelstat sodium, which includes its tautomers. Imetelstat sodium is the sodium salt of imetelstat, which is a synthetic lipid-conjugated 13-mer oligonucleotide N3’→P5’-thio-phosphoramidate. Imetelstat sodium is a telomerase inhibitor that is a covalently lipidated 13-mer oligonucleotide complementary to the human telomerase RNA (hTR) template region (shown below). The chemical name of imetelstat sodium is DNA, d(3’-amino-3’-deoxy-P-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A), 5’-[O-[2-hydroxy-3-(hexadecanoylamino)propyl] phosphorothioate], sodium salt (1:13) (SEQ ID NO: 1). Imetelstat sodium does not function through an antisense mechanism and thus has no side effects commonly observed with such therapies.

Chemical formula

[0088] Unless otherwise indicated or not clear from the context, references to imetelstat in this specification also include its tautomers and its salts, such as pharmaceutically acceptable salts. As described above, imetelstat sodium is, in particular, the sodium salt of imetelstat. Unless otherwise indicated or not clear from the context, references to imetelstat sodium in this specification also include all of its tautomers.

[0089] Imeterstat and imeterstat sodium can be manufactured, formulated, or obtained as described elsewhere (see, e.g., Asai et al., Cancer Res., 63:3931-3939 (2003), Herbert et al., Oncogene, 24:5262-5268 (2005), and Gryaznov, Chem. Biodivers., 7:477-493 (2010)). Unless otherwise indicated or not clear from the context, references to imeterstat herein include its salts. As described above, imeterstat sodium is, in particular, the sodium salt of imeterstat.

[0090] Imeterstat targets the RNA template of telomerase and inhibits telomerase activity and cell proliferation in various cancer cell lines and tumor xenografts in mice. In a Phase 1 trial of patients with breast cancer, non-small cell lung cancer and other solid tumors, multiple myeloma, or chronic lymphocytic leukemia, information regarding the pharmacokinetics and pharmacodynamics of the drug was provided. In a subsequent Phase 2 trial of patients with essential thrombocythemia, thrombocytopenic activity was shown with a significant decrease in JAK2 V617F and CALR mutant allele burden. Imeterstat sodium is administered intravenously routinely. In the practice of the subject methods, other routes of administration such as intrathecal administration, intratumoral injection, oral administration, etc. are contemplated to be usable. Imeterstat sodium can be administered at a dose equivalent to that routinely clinically utilized. In certain embodiments, imeterstat sodium is administered as described elsewhere herein.

[0091] Certain embodiments follow any one of the other embodiments, where imeterstat is limited to imeterstat sodium.

[0092] F. Pharmaceutical Compositions To facilitate administration, telomerase inhibitors (e.g., those described herein) can be formulated into various pharmaceutical forms for the purpose of administration. In some cases, the telomerase inhibitor is administered as a pharmaceutical composition. The carrier or diluent of the pharmaceutical composition must be "acceptable" in the sense that it is compatible with the other components of the composition and not harmful to its recipient. The pharmaceutical composition can be a unitary dosage form particularly suitable for oral, rectal, transdermal, parenteral injection or inhalation administration. In some cases, administration can be via intravenous injection. For example, when preparing the composition in an oral dosage form, any of the usual pharmaceutical media such as water, glycols, oils, alcohols, etc. can be employed in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions, or solid carriers such as starch, sugar, kaolin, diluents, lubricants, binders, disintegrants, etc. can be employed in the case of powders, pills, capsules and tablets. Tablets and capsules are the most advantageous oral dosage forms for easy administration, in which case a solid pharmaceutical carrier is clearly employed. For parenteral compositions, the carrier usually contains at least mostly sterile water, but may contain other components, for example, to assist solubility. For example, an injectable solution can be prepared in which the carrier contains physiological saline, a glucose solution, or a mixture of physiological saline and a glucose solution. The injectable solution containing the telomerase inhibitor described herein can be formulated in oil for long-acting effects. Suitable oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long-chain fatty acids, and mixtures of these with other oils. Injectable suspensions can be prepared, in which case suitable liquid carriers, suspending agents, etc. can be employed. Also included are solid form preparations intended to be converted to liquid form preparations immediately prior to use.In a composition suitable for transdermal administration, the carrier optionally contains a penetration enhancer and / or a suitable humectant, and is optionally combined, in small proportions, with suitable additives of any nature which do not produce any significant adverse effects on the skin. These additives can promote administration to the skin and / or can be useful for preparing the desired composition. The composition can be administered in various ways, for example, as a transdermal patch, as a spot-on, or as an ointment.

[0093] It is particularly advantageous to formulate the aforementioned pharmaceutical composition into a unit dosage form for ease of administration and uniformity of dosage. As used herein, the unit dosage form refers to physically discrete units suitable as a single dosage, each unit containing a predetermined amount of the active ingredient calculated to provide the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including divided tablets or coated tablets), capsules, pills, powder packets, wafers, suppositories, injection solutions or suspensions, etc., and multiple separate doses thereof.

[0094] To enhance the solubility and / or stability of the drugs described herein in the pharmaceutical composition, it can be advantageous to employ α-, β- or γ-cyclodextrin or their derivatives, particularly hydroxyalkyl-substituted cyclodextrin, for example, 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Also, co-solvents such as alcohol can improve the solubility and / or stability of the telomerase inhibitor in the pharmaceutical composition.

[0095] Depending on the mode of administration, the pharmaceutical composition preferably contains 0.05 to 99% by weight, more preferably 0.1 to 70% by weight, even more preferably 0.1 to 50% by weight of the telomerase inhibitor described herein, and 1 to 99.95% by weight, more preferably 30 to 99.9% by weight, even more preferably 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

[0096] G. Administration and Administration Regimen The dosing frequency can be any frequency that reduces the severity of the symptoms of myelofibrosis without causing significant toxicity to the subject. For example, the dosing frequency can be once every about 2 months to once a week, alternatively once a month to about twice a month, alternatively once every about 6 weeks, once every about 5 weeks, alternatively once every about 4 weeks, alternatively once every about 3 weeks, alternatively once every about 2 weeks, or alternatively once a week. The dosing frequency may remain constant or may vary during the course of treatment. The course of treatment with a composition containing one or more telomerase inhibitors can include a rest period. For example, a composition containing a telomerase inhibitor can be administered weekly for 3 weeks, followed by a 2-week rest period, and such a regimen can be repeated multiple times. Similar to the effective amount, various factors can affect the actual dosing frequency used for a particular application. For example, the effective amount, the treatment duration, the use of multiple therapeutic agents, the route of administration, and the severity of myelofibrosis and related symptoms may require an increase or decrease in the dosing frequency.

[0097] The effective period for administering a composition containing a telomerase inhibitor (e.g., imetelstat or imetelstat sodium) can be any period that reduces the severity of the symptoms of myelofibrosis (e.g., as described herein) without causing significant toxicity to the subject. Thus, the effective period can vary from 1 month to several months or years (e.g., 1 month to 2 years, 1 month to 1 year, 3 months to 2 years, 3 months to 10 months, or 3 months to 18 months). Generally, the effective period for the treatment of myelofibrosis can range from 2 months to 20 months. In some cases, the effective period can be as long as the individual subject lives. Multiple factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the dosing frequency, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of myelofibrosis and related symptoms.

[0098] In certain cases, the course of treatment and the severity of one or more symptoms associated with myelofibrosis can be monitored. Any method can be used to determine whether the severity of the symptoms of myelofibrosis has been reduced. For example, the severity of the symptoms of myelofibrosis (e.g., as described herein) can be evaluated using biopsy techniques.

[0099] The telomerase inhibitor used in the subject methods can be administered at any therapeutically effective dose, such as a dose equivalent to a clinically routine dose. Specific dosing regimens (e.g., recommended effective doses) for known and approved anti-cancer agents are known to physicians and are shown, for example, in the product descriptions found in PHYSICIANS’ DESK REFERENCE, 2003, 57th Ed., Medical Economics Company, Inc., Oradell, N.J., Goodman & Gilman’s THE PHARMACOLOGICAL BASIS OF THERAPEUTICS” 2001, 10th Edition, McGraw-Hill, New York, and / or are available from the Federal Drug Administration, and / or are discussed in the medical literature.

[0100] In some embodiments, the dosage of the telomerase inhibitor imetelstat sodium administered to a subject is from about 1.0 mg / kg to about 13.0 mg / kg. In other embodiments, the dosage of the telomerase inhibitor is from about 4.5 mg / kg to about 11.7 mg / kg, or from about 6.0 mg / kg to about 11.7 mg / kg, or from about 6.5 mg / kg to about 11.7 mg / kg. In some embodiments, the dosage of the telomerase inhibitor includes any one of at least about 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 10.1 mg / kg, 10.2 mg / kg, 10.3 mg / kg, 10.4 mg / kg, 10.5 mg / kg, 10.6 mg / kg, 10.7 mg / kg, 10.8 mg / kg, 10.9 mg / kg, 11 mg / kg, 11.1 mg / kg, 11.2 mg / kg, 11.3 mg / kg, 11.4 mg / kg, 11.5 mg / kg, 11.6 mg / kg, 11.7 mg / kg, 11.8 mg / kg, 11.9 mg / kg, 12 mg / kg, 12.1 mg / kg, 12.2 mg / kg, 12.3 mg / kg, 12.4 mg / kg, 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg, or 13 mg / kg.

[0101] In some embodiments, the effective amount of the telomerase inhibitor administered to the individual comprises any one of at least about 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor administered to the individual is any one of about 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 6.5 mg / kg, 7.5 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In various embodiments, the effective amount of the telomerase inhibitor administered to the individual comprises a telomerase inhibitor that is less than any one of about 350 mg / kg, 300 mg / kg, 250 mg / kg, 200 mg / kg, 150 mg / kg, 100 mg / kg, 50 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 7.5 mg / kg, 6.5 mg / kg, 5 mg / kg, 3.5 mg / kg, 2.5 mg / kg, 1 mg / kg, or 0.5 mg / kg.

[0102] Exemplary dosing frequencies of a pharmaceutical composition comprising a telomerase inhibitor include, but are not limited to, daily, every other day, twice a week, three times a week, weekly without a break, weekly, three out of four weeks, once every three weeks, once every two weeks, twice out of three weeks weekly. In some embodiments, the pharmaceutical composition is administered once about every week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. In some embodiments, the composition is administered at least once, twice, three times, four times, five times, six times, or seven times (i.e., daily) a week, or either three times a day or twice a day. In some embodiments, the interval between each administration is shorter than any of about six months, three months, one month, twenty days, fifteen days, twelve days, ten days, nine days, eight days, seven days, six days, five days, four days, three days, two days, or one day. In some embodiments, the interval between each administration is longer than any of about one month, two months, three months, four months, five months, six months, eight months, or twelve months. In some embodiments, there is no break in the dosing schedule. In some embodiments, the interval between each administration is about one week or less.

[0103] Telomerase inhibitors such as imetelstat (e.g., imetelstat sodium) can be administered using any suitable method. For example, a telomerase inhibitor such as imetelstat (e.g., imetelstat sodium) can be administered intravenously once every four weeks over a period of time (e.g., 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours). In some embodiments, imetelstat is administered intravenously once a week at 7 - 10 mg / kg over about 2 hours. In certain embodiments, imetelstat is administered intravenously once every three weeks at about 0.5 - 9.4 mg / kg over about 2 hours. In one embodiment, imetelstat is administered intravenously once every four weeks at 0.5 - 5 mg / kg over about 2 hours. In one embodiment, imetelstat is administered intravenously once every three weeks at about 2.5 - 10 mg / kg over about 2 hours. Alternatively, imetelstat is administered intravenously once every four weeks at about 0.5 - 9.4 mg / kg over about 2 hours.

[0104] In certain embodiments of the method, emetelstat is administered over 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 dosing cycles, where each cycle comprises administering emetelstat intravenously at about 7-10 mg / kg once every 3 weeks, administering emetelstat intravenously at about 7-10 mg / kg once weekly for 3 weeks, administering emetelstat intravenously at about 2.5-10 mg / kg once every 3 weeks, or administering emetelstat intravenously at about 0.5-9.4 mg / kg once every 3 weeks. In certain cases, each dosing cycle comprises administering emetelstat intravenously at about 7-10 mg / kg once every 3 weeks. In some cases, each dosing cycle comprises administering emetelstat intravenously at about 9.4 mg / kg once every about 3 weeks.

[0105] In one embodiment of the invention, emetelstat is administered intravenously at a dose of about 7-10 mg / kg once every 3 weeks after premedication with an antihistamine, a corticosteroid, or both. In other embodiments, emetelstat is administered intravenously at a dose of about 9.4 mg / kg, alternatively about 7.0 mg / kg to about 9.8 mg / kg, once every 3 weeks after premedication with an antihistamine, a corticosteroid, or both.

[0106] In certain embodiments, emetelstat is administered once every 3 weeks at a dose of about 7.5 mg / kg, alternatively about 7.0 mg / kg to about 7.7 mg / kg, for at least 3 cycles, after which the dose is increased. In certain embodiments, the dose of emetelstat may be increased to about 9.4 mg / kg, or about 8.8 mg / kg to about 9.6 kg / mg, provided that the ANC and platelet nadir are not decreased to about 1.5×10 9 / L to about 75×10 9 / L and there is no grade ≥3 non-hematological toxicity.

[0107] Cancer treatment may sometimes involve multiple "rounds" or "cycles" of drug administration, where each cycle may include one or more administrations of a drug (e.g., for 3 consecutive days every 3 weeks; once a week, etc.) according to a specified schedule. For example, an anticancer agent can be administered for 1 to 8 cycles, or for a longer period. When administering two or more drugs (e.g., two drugs) to a subject, each can be administered according to its own schedule (e.g., weekly, once every 3 weeks, etc.). It will be apparent that the administration of the drugs can be adjusted such that even if the drugs are administered in different cycles, both drugs are administered at least for some time on the same day, or alternatively, such that the drugs are administered at least for some time on consecutive days.

[0108] In certain embodiments, imetelstat can be administered via a regimen with dose reduction. In one embodiment, a patient is first administered about 9.4 mg / kg every 3 weeks, after which the dose is changed to about 7.5 mg / kg every 3 weeks, and then the dose is changed to about 6.0 mg / kg every 3 weeks.

[0109] As is understood in the art, if toxicity is observed, or for the convenience of the patient, treatment with a cancer therapeutic agent can be temporarily interrupted and then resumed without departing from the scope of the invention.

[0110] Aspects of the subject methods include identifying or selecting patients who are most likely to benefit from treatment based on the relative telomere length in a patient's target cells (e.g., as described herein). The target cells can be any convenient cells of the patient, including but not limited to cells of the patient's bone marrow or peripheral blood. In some cases, the target cells are isolated from a patient's bone marrow sample. In some cases, the target cells are isolated from a patient's peripheral blood sample. The target cells can be granulocytes. In some cases, the patient lacks mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes and has a specific short telomere length in the patient's target cells. As used herein, a short telomere length is below the median or average of the telomere length compared to a suitable control, e.g., one or more known criteria described herein. Thus, the subject methods can further include determining the relative telomere length by analyzing the relative length of telomeric nucleic acids of target cells present in a biological sample from an individual, and selecting an individual who will benefit from treatment with a telomerase inhibitor when it is determined that the average relative telomere length in the target cells present in the biological sample from the individual is at or below the 50th percentile of the relative telomere length range determined from one or more known criteria, e.g., at or below the 45th percentile, 40th percentile, 35th percentile, 30th percentile, 25th percentile, 20th percentile, or below of the relative telomere length range determined from one or more known criteria.

[0111] In some examples of the methods, the one or more known criteria are ranges of telomere lengths established from multiple naturally occurring target cells (e.g., as described herein) from multiple individuals diagnosed with the disease. In a particular example of the method, the one or more known criteria are characterized cell lines. A "characterized cell line" means that the relative telomeric nucleic acids of the cells in the cell line are known and relatively constant.

[0112] In some embodiments, the telomere length of cancer cells present in the biological sample is determined to be below the median or average telomere length. In some embodiments, the telomere length of cancer cells present in the biological sample is at or below the 50th percentile, 40th percentile, 35th percentile, 30th percentile, 25th percentile, 20th percentile, 15th percentile, 10th percentile, or 5th percentile of a relative telomere length range determined from one or more known criteria.

[0113] The telomere length of the target cells can be determined using any convenient assay including, but not limited to, qPCR, telo-FISH, or Southern blot assays as described in U.S. Patent No. 9,200,327 by Bassett et al. In one aspect, the telomere length can be determined by measuring the average length of the terminal restriction fragment (TRF). The TRF is defined as the length (usually the average length) of the fragments resulting from the complete digestion of genomic DNA by a restriction enzyme that does not cleave nucleic acids within the telomere sequence. In some cases, the DNA is digested with a restriction enzyme that cuts frequently within genomic DNA but not within the telomere sequence. In some cases, the restriction enzyme has a four-base recognition sequence (e.g., AluI, HinfI, RsaI, and Sau3A1) and is used alone or in combination. The resulting terminal restriction fragments contain both telomere repeats and subtelomeric DNA. Subtelomeric DNA is the DNA sequence adjacent to the tandem repeats of the telomere sequence and contains telomere repeat sequences interspersed with variable telomere-like sequences. The digested DNA is separated by electrophoresis and blotted onto a support such as a membrane. Fragments containing the telomere sequence are detected by hybridizing a probe, i.e., a labeled repeat sequence, to the membrane. Once the telomere-containing fragments are visualized, the average length of the terminal restriction fragments can be calculated (see Harley, C.B. et al. Nature. 345(6274):458-60(1990), incorporated herein by reference). TRF estimation by Southern blotting shows the distribution of telomere lengths within a cell or tissue and thus indicates the median and average of the telomere lengths of all cells.

[0114] In another aspect, telomere length can be measured by flow cytometry (Hultdin, M. et al., Nucleic Acids Res. 26:3651-3656 (1998), Rufer, N. et al., Nat. Biotechnol. 16:743-747 (1998), incorporated herein by reference). The flow cytometry method is a modification of the FISH technique. When the starting material is tissue, the cell suspension is generally made by mechanical dissociation and / or treatment with protease. The cells are fixed with a fixative and hybridized with a telomere sequence-specific probe labeled with a fluorescent label, preferably a PNA probe. After hybridization, the cells are washed and then analyzed by FACS. After appropriately subtracting the background fluorescence, the fluorescence signal is measured for the cells in Go / G1. This technique is suitable for rapid estimation of telomere length for a large number of samples. Similar to TRF, the telomere length is the average length of telomeres in the cell.

[0115] In other aspects, the median or average telomere length of cells from a biological sample is determined via quantitative PCR (qPCR) or telomere fluorescence in situ hybridization (telo-FISH). In qPCR, a DNA-binding dye binds to all double-stranded DNA and emits fluorescence of the dye. The increase in DNA product during the PCR reaction results in an increase in fluorescence intensity, which is measured at each cycle of the PCR reaction. This makes it possible to quantify the DNA concentration. The relative concentration of DNA present during the logarithmic phase of the reaction is determined by plotting the fluorescence level against the number of PCR cycles on a semi-log scale. A threshold value for detecting fluorescence above the background is determined. The cycle at which the fluorescence from the sample exceeds the threshold is called the cycle threshold (Ct). Since the amount of DNA theoretically doubles at each cycle during the logarithmic phase, the relative amount of DNA can be calculated. The baseline is the initial cycles of PCR where there is little change in the fluorescence signal.

[0116] In some embodiments, telomere length is determined using telo-FISH. In this method, cells are fixed and hybridized with a probe conjugated to a fluorescent label, such as Cy-3, fluorescein, rhodamine, etc. The probes for this method are oligonucleotides designed to specifically hybridize to telomere sequences. Generally, the probes are at least 8 nucleotides in length, for example at least 12-20 nucleotides in length. In one embodiment, the probe is an oligonucleotide containing naturally occurring nucleotides. In one embodiment, the probe is a peptide nucleic acid, which has a higher Tm than similar natural sequences, thus allowing the use of more stringent hybridization conditions. Cells can be treated with an agent such as colcemid to induce cell cycle arrest at metaphase and provide metaphase chromosomes for hybridization and analysis. In some embodiments, the cellular DNA can also be stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI).

[0117] A digital image of intact metaphase chromosomes is obtained and the fluorescence intensity of the probe hybridized to the telomeres is quantified. This enables measurement of the telomere length of individual chromosomes in addition to the average or median telomere length within the cell, and avoids problems associated with the presence of subtelomeric DNA (Zjilmans, J.M. et al., Proc. Natl. Acad Sci. USA 94:7423-7428 (1997), Blasco, M.A. et al. Cell 91:25-34 (1997), incorporated by reference). The intensity of the fluorescent signal correlates with the length of the telomere, and a brighter fluorescent signal indicates a longer telomere.

[0118] In certain embodiments, the invention relates to a telomerase inhibitor for use in a method of treating myelofibrosis, the method comprising identifying a patient who is most likely to benefit from treatment with a telomerase inhibitor, and administering the patient (a) Triple-negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes, (b) High molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, or (c) Both, including testing for, (a), (b), or (c) presence indicates that the patient is most likely to benefit from treatment with a telomerase inhibitor and administering an effective amount of the telomerase inhibitor to the patient. In certain embodiments, the invention relates to a telomerase inhibitor for use in a method as defined in any of the other embodiments.

[0119] Yet another embodiment of the present invention is a telomerase inhibitor for use in the treatment of myelofibrosis, the use comprising: (a) screening a patient to determine whether such patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL and / or whether such patient is at high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) administering a telomerase inhibitor to the patient if such patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL and / or is at high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes ASXL1, EZH2, SRSF2, and IDH1 / 2. In one embodiment, the use comprises screening a patient for triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL. Yet another embodiment of the present invention is a telomerase inhibitor for use in the treatment of myelofibrosis, the use comprising: (a) screening a patient to determine whether such patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL; and (b) administering a telomerase inhibitor to the patient if such patient is in a triple-negative status.

[0120] Yet another embodiment of the present invention is the use of a telomerase inhibitor for the treatment of myelofibrosis, comprising: (a) screening a patient to determine whether such a patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or whether the patient is at high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) administering a telomerase inhibitor to the patient if such a patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or if the patient is at high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes ASXL1, EZH2, SRSF2, and IDH1 / 2. In one embodiment, the use comprises screening the patient for triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL. Yet another embodiment of the present invention is the use of a telomerase inhibitor for the treatment of myelofibrosis, comprising: (a) screening a patient to determine whether such a patient is in a triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL; and (b) administering a telomerase inhibitor to the patient if such a patient is in a triple-negative status.

[0121] In certain embodiments of the present invention, the triple-negative status can be determined based on the absence of mutations in each of the JAK2 gene having the nucleic acid sequence of SEQ ID NO: 2, the CALR gene having the nucleic acid sequence of SEQ ID NO: 3, and the MPL gene having the nucleic acid sequence of SEQ ID NO: 4. In other embodiments, the triple-negative status can be determined based on the absence of mutations in each of SEQ ID NO: 2, CALR, and MPL. In alternative embodiments, the triple-negative status can be determined based on the absence of mutations in each of JAK2, SEQ ID NO: 3, and MPL. In alternative embodiments, the triple-negative status can be determined based on the absence of mutations in each of JAK2, CALR, and SEQ ID NO: 4.

[0122] In other embodiments of the present invention, the high molecular risk (HMR) can be determined based on the presence of mutations in at least one of the following genes: the ASXL1 gene having the nucleic acid sequence of SEQ ID NO: 5, the EZH2 gene having the nucleic acid sequence of SEQ ID NO: 6, the SRSF2 gene having the nucleic acid sequence of SEQ ID NO: 7, the IDH1 gene having the nucleic acid sequence of SEQ ID NO: 8, the IDH2 gene having the nucleic acid sequence of SEQ ID NO: 9, and combinations thereof.

[0123] In other embodiments of the present invention, the telomerase activity and hTERT expression levels of a biological sample obtained from a patient can be determined to evaluate the pharmacodynamic effect and / or monitor patients being treated with telomerase inhibition. Telomerase activity can be measured using the TRAP (telomere repeat amplification protocol) telomerase activity assay. The hTERT expression level can be determined by measuring the hTERT RNA expression level in cells in the biological sample using Northern blot or serial analysis of gene expression (SAGE) or other methods.

[0124] In certain embodiments, the present invention relates to a telomerase inhibitor for use in the treatment of myelofibrosis as defined in any of the other embodiments.

[0125] In certain embodiments, the invention relates to the use of a telomerase inhibitor for the treatment of myelofibrosis as defined in any of the other embodiments.

[0126] Additional embodiments Additional embodiments of the object are described in the following clauses:

[0127] Clause 1. A method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) Examining a patient for triple-negative status based on the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes; and (b) Selecting a patient if the patient has triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, wherein the selected patient is most likely to benefit from treatment with a telomerase inhibitor.

[0128] Clause 2. A method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (c) Examining a patient for: i. Triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. High molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (d) Selecting a patient having: i. Triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. High molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. A method by which the selected patient is most likely to benefit from treatment with a telomerase inhibitor.

[0129] Clause 3. A method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (e) obtaining a DNA sample from the patient; and (f) testing the DNA sample derived from the patient for a triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and (g) selecting the patient if the patient has a triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes. A method by which the selected patient is most likely to benefit from treatment with a telomerase inhibitor.

[0130] Clause 4. A method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, comprising: (h) obtaining a DNA sample from the patient; and (i) testing the DNA sample derived from the patient for: i. a triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. a high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (j) selecting the patient if the patient has: i. a triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. a high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. A method by which the selected patient is most likely to benefit from treatment with a telomerase inhibitor.

[0131] Item 5. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, where the patient is determined to have a triple-negative status, wherein the triple-negative status comprises no mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. Use of the telomerase inhibitor.

[0132] Item 6. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, where the patient is determined to have a high molecular risk (HMR), wherein having HMR comprises the presence of a mutation in at least one gene selected from the group consisting of additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2). Use of the telomerase inhibitor.

[0133] Item 7. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, where it has been determined that the cells present in a patient-derived biological sample have an average relative telomere length that is at or below the 50th percentile of a range of relative telomere lengths determined from one or more known criteria. Use of the telomerase inhibitor.

[0134] Item 8. Use of a telomerase inhibitor in the manufacture of a medicament for the treatment of a patient with myelofibrosis, where the patient is determined to have a triple-negative status,

[0135] Use of a telomerase inhibitor in the manufacture of a medicament for the treatment of a patient having myelofibrosis, wherein the patient is determined to have a high molecular risk (HMR), the presence of a mutation in at least one gene selected from the group consisting of additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2) being included in having HMR.

[0136] Use of a telomerase inhibitor in the manufacture of a medicament for the treatment of a patient having myelofibrosis, wherein the cells present in a biological sample derived from the patient are determined to have an average relative telomere length that is at or below the 50th percentile of the range of relative telomere lengths determined from one or more known criteria.

[0137] The following examples are provided by way of illustration and not limitation.

Example

[0138] Example 1: Imetelstat sodium is an effective treatment for patients having intermediate-2 (int-2) or high-risk myelofibrosis (MF) that has relapsed or is resistant to Janus kinase (JAK) inhibitor therapy. Introduction Imetelstat, a 13-mer oligonucleotide that specifically targets the RNA template of human telomerase, is a potent competitive inhibitor of telomerase enzyme activity (Asai et al. Cancer Res 2003; Herbert, Oncogene 2005). Clinical activity and an acceptable safety profile were reported in a pilot trial of 33 patients with intermediate-2 (int-2) or high-risk myelofibrosis (MF), 48% of whom had been previously treated with a Janus kinase inhibitor (JAKi) (Tefferi, N Engl J Med 2015). This example provides the results of a phase 2 clinical trial of imetelstat sodium at two dose levels in patients with myelofibrosis (MF).

[0139] Methods A randomized, multi-center, phase 2 trial of twice-dosed imetelstat sodium (9.4 mg / kg or 4.7 mg / kg IV once every 3 weeks) was conducted in adults with a Dynamic International Prognostic Scoring (DIPSS) score of int-2 or high-risk MF who were refractory / relapsed to prior JAKi therapy (i.e., either no reduction in splenomegaly after 12 weeks or worsening of splenomegaly at any point after initiation of JAK inhibitor (「JAKi」) therapy). A diagnosis of MF after polycythemia vera, essential thrombocythemia or primary myelofibrosis was required. Other eligibility criteria included measurable splenomegaly (by magnetic resonance imaging [MRI]), active MF-related constitutional symptoms, and platelet count ≥75 × 10 9 / L. The primary endpoints were the spleen response rate (percentage achieving ≥35% reduction in spleen volume [SVR] by MRI at week 24) and the symptom response rate (percentage achieving ≥50% reduction in total symptom score [TSS] according to the Myelofibrosis Symptom Assessment Form (MFSAF) v2 at week 24). Secondary endpoints included safety, overall survival (OS), treatment response, molecular response, and pharmacokinetic and pharmacodynamic relationships.

[0140] Results A total of 107 patients were enrolled at 55 sites (48 patients for 4.7 mg / kg and 59 patients for 9.4 mg / kg). Baseline characteristics are shown in Table 1 below. Additionally, the median time on JAKi was 23 months (0.9 - 89.7), and the median platelet count was 147×10 9 / L. Triple negative (TN, i.e., no mutations in JAK2, MPL, or CALR) accounted for 24.8% of the patients, and 67.6% were considered high molecular risk (HMR, i.e., ≥1 mutation in ASXL1, EZH2, SRSF2, or IDH1 / 2). [Table 1]

[0141] At the time of the primary clinical cutoff, the median time of the trial was 22.6 months (range, 0.2 - 27.4 months), and the median treatment time was 6.2 months (range, 0.0 - 27.2 months). Six (10.2%) patients in the 9.4 mg / kg group had a spleen response per MRI confirmed by the IRC

[0142] At the time of the clinical cutoff, patients had a median follow-up of 22.6 (0.2 - 27.4) months, and the median treatment duration was 6.2 (0.0 - 27.2) months. The median treatment duration was longer in the 9.4 mg / kg group (7.7 months) than in the 4.7 mg / kg group. Six (10.2%) patients in the 9.4 mg / kg group had a spleen response per MRI, and there was no response in the 4.7 mg / kg group (see Figure 1). Nineteen (32%) patients in the 9.4 mg / kg group and three (6%) patients in the 4.7 mg / kg group had a symptomatic response (TSS reduction ≥50%) (see Figure 2).

[0143] At the initial clinical cutoff, the median OS in the 9.4 mg / kg group had not been reached, while the median OS in the 4.7 mg / kg group was 19.9 months. The 18-month survival rates in the 9.4 mg / kg and 4.7 mg / kg groups were 76.7% and 62.9%, respectively. In the sensitivity analysis, similar results occurred by censoring patients during subsequent dose escalations for JAKi therapy or stem cell transplantation. In the 9.4 mg / kg group, a correlation was observed between TN patients and OS patients (the median OS was not reached in TN patients and was 23.6 months in non-TN patients). The spleen response rate was higher in patients with one HMR mutation (ASXL1, EZH2, SRSF2, or IDH1 / 2).

[0144] The most common adverse events (all grades) with treatment at 9.4 mg / kg were thrombocytopenia (49%), anemia (44%), neutropenia (36%), and nausea (34%), and at 4.7 mg / kg were diarrhea (38%), nausea (31%), anemia (31%), and thrombocytopenia (23%). Grade 3 / 4 neutropenia and thrombocytopenia were more frequent at 9.4 mg / kg (34% and 42%, respectively) than at 4.7 mg / kg (13% and 29%, respectively), and most cytopenias resolved within 4 weeks. Grade 3 / 4 LFT elevations were observed in 7 patients during the trial. No imetelstat-related hepatotoxicity was confirmed by the independent Hepatic Review Committee.

[0145] At the time of the second clinical cutoff, patients were followed up for 27.4 (0.2 - 33.0) months, and the median treatment period was 26.9 (0.1 - 118.1) weeks. The median treatment period was longer in the 9.4 mg / kg group (33.3 weeks) than in the 4.7 mg / kg group (23.9 weeks). Early closure of the 4.7 mg / kg group affected the treatment period. The median OS with 95% confidence interval in the 9.4 mg / kg group reached 29.9 months (22.8, NE) (NE is not estimable) in the 9.4 mg / kg group at the second clinical cutoff.

[0146] Triple-negative vs OS Subjects are grouped by the mutation status of the JAK2 / MPL / CALR genes, triple-negative (TN, no mutation in each of the JAK2 / MPL / CALR genes), and non-TN (having a mutation in any of the JAK2 / MPL / CALR genes). In the 9.4 mg / kg group, the median OS could not be estimated (NE) for TN subjects with a 95% confidence interval (23.2, NE), and was 23.6 months for non-TN subjects with a 95% confidence interval (20.7, NE), while at 4.7 mg / kg, the median OS with 95% confidence was 22.3 (17, NE) and interval 20.3 (18.3, NE) for TN and non-TN subjects, respectively. In the 9.4 mg / kg group, in the triple-negative (TN) group, a lower mortality was observed compared to the non-TN group (see Table 2, Figures 3 and 4).

Table 2

[0147] At the second clinical cut-off, in the triple-negative (TN) group, a lower mortality was observed in the 9.4 mg / kg group compared to the non-TN group (see Table 3, Figures 6 and 7).

Table 3

[0148] Triple-negative vs response at week 24 In the 9.4 mg / kg group, a higher response rate (SVR or TSS) was observed in the TN group compared to the non-TN group (see Table 4 below).

Table 4

[0149] Molecular risk vs response at week 24 In the 9.4 mg / kg group, higher response rates (SVR or TSS) were observed in the low molecular risk (LMR) group or the high molecular risk (HMR) group with only one mutation (mut) compared to the HMR group with more than one mutation (see Table 5).

Table 5

[0150] For the 9.4 mg / kg subjects, an association was observed between the following factors and clinical response or OS. Triple negative (TN): Response (SVR or TSS) was enhanced in TN subjects. Median OS could not be estimated for TN, non-TN = 23.6 months, and Molecular risk: Response (SVR or TSS) was enhanced in subjects with HMR with only one mutation, and response was observed in patients with HMR with more than one mutation treated with 9.4 mg / kg of imetelstat.

[0151] Example 2 Baseline telomere length (TL) vs overall survival (OS) Subjects were grouped by the median of baseline TL. In the 9.4 mg / kg group, the median OS could not be estimated (NE) at the first clinical cut-off, and was (23.2, NE) in the 95% confidence interval for subjects with shorter baseline TL (<= median) and 22.8 (16.2, NE) months for subjects with longer TL (> median), respectively (Table 6). In the 4.7 mg / kg group, the median OS by 95% confidence interval was 20.3 (17.2, NE) months and 22.3 (16.6, NE) months for subjects with shorter and longer baseline TL, respectively (Table 6).

[0152] In the 9.4 mg / kg group, a better OS trend was observed for subjects with shorter baseline TL, i.e., subjects with baseline TL below the median TL.

Table 6

[0153] Response to baseline TL at week 24 Baseline telomere length (TL): The SVR or TSS response at week 24 was enhanced in subjects with shorter baseline TL (<= median). 17.3% (5 / 29) of subjects with shorter baseline TL and 4.2% (1 / 24) of subjects with longer baseline TL had spleen responses, respectively. 34.5% (10 / 29) of subjects with shorter baseline TL and 25% (6 / 24) of subjects with longer baseline TL had TSS responses, respectively.

[0154] In the 9.4 mg / kg group, at week 24, in subjects with shorter baseline TL, a higher response rate (SVR or TSS) was enhanced compared to subjects with longer TL (Table 7).

Table 7

[0155] Example 3 Dose-dependent pharmacodynamic (PD) effects Telomerase activity and hTERT were analyzed to evaluate the pharmacodynamic effects of imetelstat. Among subjects with available baseline and post-treatment data, 23 (51.1%) subjects in the 9.4 mg / kg group and 10 (29.4%) subjects in the 4.7 mg / kg group achieved >= 50% reduction in telomerase activity from baseline, which is a PD effect and showed a correlation with anti-tumor activity from preclinical xenograft models in vivo. In addition, 35 (61.4%) subjects in the 9.4 mg / kg group and 20 (47.7%) subjects in the 4.7 mg / kg group achieved >= 50% reduction in hTERT RNA levels from baseline, respectively (Table 8). Therefore, a dose-dependent PD effect was demonstrated and target engagement was shown.

Table 8

[0156] Example 4 Association between the PD effect and response at week 24 Subjects who were spleen responders at a higher rate (83.3%) achieved at least a >=50% decrease in hTERT RNA expression levels compared to subjects who were non-spleen responders (55.6%), and subjects who had a higher rate of TSS response achieved at least a >=50% decrease in hTERT RNA expression levels compared to non-TSS responders (Table 9). The hTERT RNA expression levels were measured from whole blood samples collected from patients before and after treatment. [Table 9]

[0157] Subjects with a higher rate of spleen response or TSS response achieved at least a >=30% or >=50% decrease in telomerase activity compared to subjects without spleen response or TSS response (Table 10). [Table 10]

[0158] Aspects, including embodiments, of the subject matter described herein may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the description, certain non-limiting aspects of the present disclosure are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below. 1. Use of a telomerase inhibitor in the treatment of a patient having myelofibrosis, wherein the patient is determined to have a triple-negative status, The triple-negative status includes no mutation in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. 2. The use according to embodiment 1, wherein the myelofibrosis is primary myelofibrosis. 3. The use according to embodiment 2, wherein the myelofibrosis is myelofibrosis developing after polycythemia vera (post-PV MF). 4. The use according to embodiment 2, wherein the myelofibrosis is myelofibrosis developing after essential thrombocythemia (post-ET MF). 5. The use according to any one of embodiments 1 to 4, wherein the patient has never received JAK inhibitor therapy before. 6. The use according to any one of claims 1 to 4, wherein the patient has received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 7. The use according to any one of embodiments 1 to 4, wherein the patient has received JAK inhibitor therapy and has relapsed. 8. The use according to any one of embodiments 1 to 4, wherein the patient has received JAK inhibitor therapy and the JAK inhibitor therapy was discontinued due to treatment-related toxicity or intolerance. 9. The use according to any one of embodiments 1 to 8, wherein the telomerase inhibitor is imetelstat. 10. The use according to embodiment 9, wherein imetelstat is imetelstat sodium. 11. The telomerase inhibitor is imetelstat and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising intravenously administering about 7 - 10 mg / kg of imetelstat once every 3 weeks, intravenously administering about 7 - 10 mg / kg of imetelstat once weekly for 3 weeks, intravenously administering about 2.5 - 10 mg / kg of imetelstat once every 3 weeks, or intravenously administering about 0.5 - 9.4 mg / kg of imetelstat once every 3 weeks, the use according to embodiment 10. 12. The use according to embodiment 11, wherein each dosing cycle comprises intravenously administering about 7 - 10 mg / kg of imetelstat once every 3 weeks. 13. The use according to embodiment 12, wherein each dosing cycle comprises intravenous administration of about 9.4 mg / kg of imetelstat once every three weeks. 14. The use according to any one of embodiments 1 to 13, wherein the average relative telomere length is determined by analyzing the relative length of telomeric nucleic acids in target cells present in a patient-derived biological sample. 15. The use according to any one of embodiments 1 to 14, further comprising selecting a patient identified as having an average relative telomere length in target cells present in a patient-derived biological sample, which is determined to be at or below the 50th percentile of a relative telomere length range determined from one or more known criteria. 16. The use according to any one of embodiments 1 to 15, further comprising screening a patient to determine whether the patient has high molecular risk (HMR), wherein having HMR includes the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2, and IDH1 / 2. 17. The use according to any one of embodiments 1 to 16, further comprising evaluating the hTERT expression level in a biological sample obtained from a patient after administration of a telomerase inhibitor. 18. The use according to embodiment 17, wherein the hTERT expression level is reduced by 50% or more compared to the baseline hTERT expression level before administration of the telomerase inhibitor. 19. The use according to embodiment 17 or 18, further comprising changing the dosage, number of administrations, or treatment course administered to a subject of the telomerase inhibitor. 20. Use of a telomerase inhibitor in the treatment of a patient having myelofibrosis, wherein the patient is determined to have high molecular risk (HMR), and having HMR includes the presence of a mutation in at least one gene selected from the group consisting of additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2). 21. The use according to embodiment 20, wherein the myelofibrosis is primary myelofibrosis. 22. The use according to embodiment 21, wherein the myelofibrosis is myelofibrosis developing after polycythemia vera (PV-post MF). 23. The use according to embodiment 21, wherein the myelofibrosis is myelofibrosis developing after essential thrombocythemia (ET-post MF). 24. The use according to any one of embodiments 20 - 23, wherein the patient has never received JAK inhibitor therapy before. 25. The use according to any one of embodiments 20 - 23, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 26. The use according to any one of embodiments 20 - 23, wherein the patient has received JAK inhibitor therapy and has relapsed. 27. The use according to any one of embodiments 20 - 23, wherein the patient has received JAK inhibitor therapy and the JAK inhibitor therapy has been discontinued due to treatment-related toxicity or intolerance. 28. The use according to any one of embodiments 20 - 27, wherein the telomerase inhibitor is imetelstat. 29. The use according to embodiment 28, wherein imetelstat is imetelstat sodium. 30. The telomerase inhibitor is imetelstat and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, and each cycle comprises intravenously administering about 7 - 10 mg / kg of imetelstat once every 3 weeks, intravenously administering about 7 - 10 mg / kg of imetelstat once a week for 3 weeks, intravenously administering about 2.5 - 10 mg / kg of imetelstat once every 3 weeks, or intravenously administering about 0.5 - 9.4 mg / kg of imetelstat once every 3 weeks, the use according to embodiment 28. 31. The use according to embodiment 30, wherein each dosing cycle comprises intravenously administering about 7 - 10 mg / kg of imetelstat once every 3 weeks. 32. The use according to embodiment 31, wherein each dosing cycle comprises intravenously administering about 9.4 mg / kg of imetelstat once every 3 weeks. 33. The use according to any one of aspects 20 to 32, further comprising determining the average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a patient-derived biological sample. 34. The use according to any one of aspects 20 to 33, further comprising selecting a patient identified as having an average relative telomere length in target cells present in a patient-derived biological sample that is determined to be below the 50th percentile of a relative telomere length range determined from one or more known criteria. 35. The use according to any one of aspects 20 to 34, further comprising screening the patient to determine whether the patient is in a triple-negative status, wherein the triple-negative status includes no mutation in each of the genes selected from the group consisting of JAK2, CALR, and MPL. 36. The use according to any one of aspects 20 to 35, further comprising evaluating the hTERT expression level in a biological sample obtained from the patient after administration of a telomerase inhibitor. 37. The use according to aspect 36, wherein the hTERT expression level is reduced by 50% or more compared to the baseline hTERT expression level before administration of the telomerase inhibitor. 38. The use according to any one of aspects 36 to 37, further comprising changing the dosage, frequency of administration, or treatment course administered to the subject of the telomerase inhibitor. 39. The use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the cells present in the patient-derived biological sample are determined to have an average relative telomere length that is below the 50th percentile of a relative telomere length range determined from one or more known criteria. 40. The use according to aspect 39, wherein the myelofibrosis is primary myelofibrosis. 41. The use according to aspect 40, wherein the myelofibrosis is myelofibrosis developing after polycythemia vera (PV post-MF). 42. The use according to aspect 40, wherein the myelofibrosis is myelofibrosis developing after essential thrombocythemia (ET post-MF). 43. Use according to any one of aspects 39 to 42, wherein the patient has not previously received JAK inhibitor therapy. 44. Use according to any one of aspects 39 to 42, wherein the patient has received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 45. Use according to any one of aspects 39 to 42, wherein the patient has received JAK inhibitor therapy and has relapsed. 46. Use according to any one of aspects 39 to 42, wherein the patient has received JAK inhibitor therapy and the JAK inhibitor therapy was discontinued due to treatment-related toxicity or intolerance. 47. Use according to any one of aspects 39 to 46, wherein the telomerase inhibitor is imetelstat. 48. Use according to aspect 47, wherein imetelstat is imetelstat sodium. 49. Use according to aspect 47, wherein the telomerase inhibitor is imetelstat and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, and each cycle comprises intravenously administering about 7 - 10 mg / kg of imetelstat once every 3 weeks, intravenously administering about 7 - 10 mg / kg of imetelstat once weekly for 3 weeks, intravenously administering about 2.5 - 10 mg / kg of imetelstat once every 3 weeks, or intravenously administering about 0.5 - 9.4 mg / kg of imetelstat once every 3 weeks. 50. Use according to aspect 49, wherein each dosing cycle comprises intravenously administering about 7 - 10 mg / kg of imetelstat once every 3 weeks. 51. Use according to aspect 50, wherein each dosing cycle comprises intravenously administering about 9.4 mg / kg of imetelstat once every 3 weeks. 52. Use according to any one of aspects 39 to 51, further comprising determining the average relative telomere length by analyzing the relative length of telomeric nucleic acids in cells present in a biological sample derived from the patient. The use according to any one of aspects 39 to 52, further comprising evaluating the hTERT expression level in a biological sample obtained from a patient after administration of a telomerase inhibitor. The use according to aspect 53, wherein the hTERT expression level is decreased by 50% or more compared to the baseline hTERT expression level before administration of the telomerase inhibitor. The use according to any one of aspects 53 to 54, further comprising changing the dosage, the number of administrations, or the treatment course administered to the subject of the telomerase inhibitor. A method for selecting a patient who is most likely to benefit from treatment with a telomerase inhibitor, comprising: examining the patient for a triple negative status, wherein the triple negative status includes no mutations in each of the JAK2, CALR, and MPL genes, and selecting the patient if the patient has a triple negative status, wherein the selected patient is most likely to benefit from treatment with a telomerase inhibitor. The method according to aspect 56, wherein the patient has myelofibrosis. The method according to aspect 57, wherein the myelofibrosis is primary myelofibrosis. The method according to aspect 57, wherein the myelofibrosis is myelofibrosis developing after polycythemia vera (post-PV MF). The method according to aspect 57, wherein the myelofibrosis is myelofibrosis developing after essential thrombocythemia (post-ET MF). The method according to any one of aspects 56 to 60, wherein the patient has never received JAK inhibitor therapy before. The patient has previously received JAK inhibitor therapy, previously received JAK inhibitor therapy and has failed JAK inhibitor therapy; or previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. The method according to any one of aspects 56 to 60. 63. The method according to any one of aspects 56 to 60, wherein the patient has received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 64. The method according to any one of aspects 56 to 60, wherein the patient has received JAK inhibitor therapy and has relapsed. 65. The method according to any one of aspects 56 to 60, wherein the patient has received JAK inhibitor therapy and the JAK inhibitor therapy was discontinued due to treatment-related toxicity or intolerance. 66. The method according to any one of aspects 56 to 65, further comprising administering a telomerase inhibitor to the patient. 67. The method according to aspect 66, wherein the telomerase inhibitor is imetelstat. 68. The method according to aspect 67, wherein the imetelstat is imetelstat sodium. 69. The method according to any one of aspects 56 to 68, further comprising obtaining a sample containing DNA from the patient. 70. The method according to aspect 69, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 71. The step of obtaining a sample from the patient is obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof, and isolating DNA from the bone marrow sample, the peripheral blood sample, or a combination thereof, the method according to aspect 70. 72. The step of obtaining a sample from the patient is obtaining a bone marrow sample from the patient, and isolating cells from the bone marrow sample, and extracting DNA from the isolated cells, the method according to aspect 70. 73. The step of obtaining a sample from the patient is obtaining a peripheral blood sample from the patient, and isolating cells from the peripheral blood sample, and extracting DNA from the isolated cells, the method according to aspect 70. 74. A method for selecting a patient who is most likely to benefit from treatment with a telomerase inhibitor, Examining a patient to determine whether the patient has HMR, wherein having HMR includes the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2, and IDH1 / 2, and selecting the patient if the patient has HMR, wherein the selected patient is most likely to benefit from treatment with a telomerase inhibitor. 75. The method according to embodiment 74, wherein the patient has myelofibrosis. 76. The method according to embodiment 75, wherein the myelofibrosis is primary myelofibrosis. 77. The method according to embodiment 75, wherein the myelofibrosis is myelofibrosis developing after polycythemia vera (post-PV MF). 78. The method according to embodiment 75, wherein the myelofibrosis is myelofibrosis developing after essential thrombocythemia (post-ET MF). 79. The method according to any one of embodiments 74 to 78, wherein the patient has not previously received JAK inhibitor therapy. 80. The patient is has previously received JAK inhibitor therapy, has previously received JAK inhibitor therapy and has failed JAK inhibitor therapy, or has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance, according to any one of embodiments 74 to 78. 81. The method according to any one of embodiments 74 to 78, wherein the patient has received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 82. The method according to any one of embodiments 74 to 78, wherein the patient has received JAK inhibitor therapy and has relapsed. 83. The method according to any one of embodiments 74 to 78, wherein the patient has received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 84. The method according to any one of embodiments 74 to 83, further comprising administering a telomerase inhibitor to the patient. The method according to embodiment 84, wherein the telomerase inhibitor is imetelstat. The method according to embodiment 85, wherein the imetelstat is imetelstat sodium. The method according to any one of embodiments 74 to 86, further comprising obtaining a sample containing DNA from a patient. The method according to embodiment 87, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 89. The step of obtaining a sample from a patient comprises obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof, and isolating DNA from the bone marrow sample, the peripheral blood sample, or a combination thereof, the method according to embodiment 88. 90. The step of obtaining a sample from a patient comprises obtaining a bone marrow sample from the patient, isolating cells from the bone marrow sample, and extracting DNA from the isolated cells, the method according to embodiment 88. 91. The step of obtaining a sample from a patient comprises obtaining a peripheral blood sample from the patient, isolating cells from the peripheral blood sample, and extracting DNA from the isolated cells, the method according to embodiment 88. 92. A method for selecting a patient who is most likely to benefit from treatment with a telomerase inhibitor, comprising examining the patient for mean relative telomere length by analyzing the relative length of telomeric nucleic acids of target cells present in a biological sample derived from the patient, and selecting the patient if the patient has a mean relative telomere length of target cells present in a biological sample derived from the patient that is determined to be below the 50th percentile of a relative telomere length range determined from one or more known criteria, wherein the selected patient is most likely to benefit from treatment with a telomerase inhibitor. 93. The method according to embodiment 92, wherein the patient has myelofibrosis. 94. The method according to embodiment 93, wherein the myelofibrosis is primary myelofibrosis. 95. The method according to embodiment 93, wherein the myelofibrosis is myelofibrosis developing after polycythemia vera (post-PV MF). 96. The method according to embodiment 93, wherein the myelofibrosis is myelofibrosis developing after essential thrombocythemia (post-ET MF). 97. The method according to any one of embodiments 92-96, wherein the patient has not previously received JAK inhibitor therapy. 98. The patient has previously received JAK inhibitor therapy, has previously received JAK inhibitor therapy and has failed JAK inhibitor therapy; or has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance, according to any one of embodiments 92-96. 99. The method according to any one of embodiments 92-96, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 100. The method according to any one of embodiments 92-96, wherein the patient has received JAK inhibitor therapy and has relapsed. 101. The method according to any one of embodiments 92-96, wherein the patient has received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 102. The method according to any one of embodiments 92-101, further comprising administering a telomerase inhibitor to the patient. 103. The method according to embodiment 102, wherein the telomerase inhibitor is imetelstat. 104. The method according to embodiment 103, wherein imetelstat is imetelstat sodium. 105. The method according to any one of embodiments 92-104, further comprising obtaining a sample containing DNA from the patient. 106. The method according to embodiment 105, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 107. The step of obtaining a sample from the patient obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof, isolating DNA from a bone marrow sample, a peripheral blood sample, or a combination thereof, the method according to embodiment 106. 108. The step of obtaining a sample from a patient is obtaining a bone marrow sample from the patient, isolating cells from the bone marrow sample, extracting DNA from the isolated cells, the method according to embodiment 106. 109. The step of obtaining a sample from a patient is obtaining a peripheral blood sample from the patient, isolating cells from the peripheral blood sample, extracting DNA from the isolated cells, the method according to embodiment 106. 110. A method for monitoring therapeutic efficacy in a subject having myelofibrosis (MF), the method comprising measuring the hTERT expression level in a biological sample obtained from a patient after administration of a telomerase inhibitor, comparing the hTERT expression level in the biological sample with the baseline hTERT expression level before administration of the telomerase inhibitor, a decrease of 50% or more in the hTERT expression level in the biological sample identifies a subject likely to benefit from treatment with a telomerase inhibitor. 111. The method according to embodiment 110, wherein the hTERT expression level to be measured or evaluated is the hTERT RNA expression level. 112. A method for identifying a patient having myelofibrosis (MF) for treatment with a telomerase inhibitor, the method comprising measuring the hTERT expression level in a biological sample obtained from a patient after administration of a telomerase inhibitor, comparing the hTERT expression level in the biological sample with the baseline hTERT expression level before administration of the telomerase inhibitor, a decrease in the hTERT expression level in the biological sample identifies a patient likely to benefit from treatment with a telomerase inhibitor. 113. The method according to embodiment 112, wherein the decrease in the hTERT expression level is 50% or more.

[0159] Certain embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, but it will be readily apparent that, in light of the teachings of the present invention, certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0160] Accordingly, the foregoing is only illustrative of the principles of the invention. Although not explicitly described or shown herein, various combinations that embody the principles of the invention and fall within its spirit and scope may be devised. Further, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Additionally, all descriptions in this specification listing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, regardless of structure, i.e., any elements developed to perform the same function. Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied by the appended claims.

Claims

【Claim 1】 The invention described in the drawings.

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