How to identify patients who are likely to benefit from telomerase inhibitor treatment

Abstract

For example, to provide a method for identifying or selecting patients who are most likely to benefit from treatment with telomerase inhibitors such as imetelstat. [Solution] This disclosure provides a method for identifying or selecting patients who are most likely to benefit from treatment with telomerase inhibitors, such as imetelstat, by testing for high molecular weight risk (HMR) based on the absence of mutations in each of JAK2, CALR, and MPL, and / or the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. These patients may have myelofibrosis. This disclosure also provides a method for treating myelofibrosis, the method of which includes identifying such patients.

Description

Technical Field

[0001] Cross - Reference to Related Applications Under 35 U.S.C.§119(e), 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, the disclosures of which 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 classic BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by clonal myeloproliferation and dysregulated kinase signaling. Cervantes, Blood, 124(17):2635-2642 (2014). It is also characterized by cytopenia, systemic symptoms, and splenomegaly, and can 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 the JAK1 / JAK2 inhibitor ruxolitinib is the currently approved treatment. Ruxolitinib, a Janus kinase (JAK)-1 and JAK-2 inhibitor, is a first-in-class drug licensed in the United States for the treatment of high-risk and moderate-risk myelofibrosis (MF). Pardanani, et al.; Blood Cancer J.; 4(12):e268(2014). Several other JAK inhibitors are under development, some currently in Phase 3 clinical trials. Ibid. Other treatment options for MF include allo-SCT, hydroxyurea, interferon, lenalidomide (Revlimid®), and thalidomide. There are currently 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. al., Expert Opin Emerg Drugs, 23(1):37-49(2018).

[0005] Other MPNs include essential thrombocythemia (ET) and polycythemia vera (PV). Cervantes (see below). MF can appear newly (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 pluripotent hematopoietic stem cells, and the abnormal cell population releases several cytokines and growth factors into the bone marrow, causing myelofibrosis and stromal changes, and colonizing extramedullary organs such as the spleen and liver. Ibid. Myelofibrosis has been 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 cure other than allogeneic hematopoietic stem cell transplantation (allo-SCT), which can be applied to a small number of patients." Ibid.

[0006] In fact, according to Langabeer, "the vast majority of patients with classical myeloproliferative neoplasms (MPNs) such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis have a distinct disease-driving mutation in the JAK2, CALR, or MPL gene." Langabeer, JAK-STAT, 5:e1248011 (2016). These mutations are so-called driver mutations. Exemplary driver mutations include those 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 stage, and carry the greatest risk conferred by triple-negative status (i.e., non-mutated 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 most unfavorable outcome. 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 weight risk (HMR) genes such as ASXL1, EZH2, IDH1 / 2, and SRSF2 are also associated with poor prognosis. Spiegel et al. The presence of an increasing number of prognostically harmful / "high molecular risk" mutations (i.e., ASXL1, EZH2, SRSF2, and / or IDH-1 / 2 genes) led to progressively worse survival outcomes, independently of conventional risk factors. Guglielmelli et al., Leukemia, 28(9):1804-10 (2014).

[0008] Driver mutations in JAK2, MPL, or CALR, either alone or in combination with subclonal mutations in genes such as ASXL1, are associated with differences in overall survival (OS). 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 these mutations were associated with a shorter time to treatment failure in patients with myelofibrosis treated with ruxolitinib or momerotinib (JAK1 / 2 inhibitors). Ibid. Similarly, "When 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), trends reported in other MPN cohorts." Patel et al., Blood;126(6):790-797 (2015). Patel et al. observed that patients treated with ruxolitinib with three or more mutations showed an inverse correlation between splenic response and time to treatment discontinuation. Driver mutations or triple-negative (JAK2, MPL, CALR) status are found in myelofibrosis patients discontinuing JAK inhibitor treatment. See, for example, Kuykendall et al. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Cervantes,Blood,124(17):2635-2642(2014) [Overview of the Initiative] [Means for solving the problem]

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

[0011] One embodiment of the present invention is a method for identifying myelofibrosis patients 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 following genes: (i) 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 a patient if the patient has triple-negative status based on the absence of mutations in each of the following genes, and / or (ii) high molecular weight 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 patient is most likely to benefit from treatment with a telomerase inhibitor.

[0012] Alternative embodiments of the present invention are methods for identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising (a) testing patients for triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and (b) selecting patients if they have triple-negative status, the selected patients being most likely to benefit from treatment with a telomerase inhibitor. Alternative embodiments of the present invention are methods for identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising (a) testing patients for high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, and (b) selecting patients with high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The present invention further provides methods for treating myelofibrosis in patients with triple-negative and / or HMR 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) examining the DNA sample from such patient for triple-negative status based on (i) 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 who has triple-negative status based on (i) the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or 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 patient is 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] A DNA sample can be obtained by first acquiring a bone marrow sample, a peripheral blood sample, or both, and then isolating the 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 includes 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 includes 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] A further 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 testing the patients for (a) triple-negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes, (b) high molecular weight 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 thereof, 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 may be primary myelofibrosis, myelofibrosis that develops after polycythemia vera (post-PV MF), or myelofibrosis that develops after essential thrombocythemia (post-ET MF). In certain embodiments, the patient has never 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 relapsed). In other embodiments, the patient has received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

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

[0018] When using imeterstat to treat patients identified by these methods, imeterstat is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 administration cycles, each cycle comprising intravenous administration of approximately 7–10 mg / kg of imeterstat once every 3 weeks, intravenous administration of approximately 7–10 mg / kg of imeterstat once a week for 3 weeks, intravenous administration of approximately 2.5–10 mg / kg of imeterstat once every 3 weeks, or intravenous administration of approximately 0.5–9.4 mg / kg of imeterstat once every 3 weeks. In one embodiment, each administration cycle comprises intravenous administration of approximately 7–10 mg / kg of imeterstat once every 3 weeks. In another embodiment, each administration cycle comprises intravenous administration of approximately 9.4 mg / kg of imeterstat once every 3 weeks.

[0019] When using imeterstat sodium to treat patients identified by these methods, imeterstat sodium is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 administration cycles, each cycle comprising intravenous administration of approximately 7–10 mg / kg of imeterstat sodium once every 3 weeks, intravenous administration of approximately 7–10 mg / kg of imeterstat sodium once a week for 3 weeks, intravenous administration of approximately 2.5–10 mg / kg of imeterstat sodium once every 3 weeks, or intravenous administration of approximately 0.5–9.4 mg / kg of imeterstat sodium once every 3 weeks. In one embodiment, each administration cycle comprises intravenous administration of approximately 7–10 mg / kg of imeterstat sodium once every 3 weeks. In another embodiment, each administration cycle comprises intravenous administration of approximately 9.4 mg / kg of imeterstat sodium once every 3 weeks.

[0020] Another embodiment of the present invention is a method for treating a patient with myelofibrosis with a telomerase inhibitor such as imeterstat or imeterstat sodium, (i) Screening patients to determine whether they are triple-negative based on the absence of mutations in each of JAK2, CALR, and MPL, and / or whether they are at high risk for high molecular weight (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, (ii) A method comprising administering a telomerase inhibitor to such a patient if the patient is triple-negative based on the absence of mutations in any of JAK2, CALR, and MPL, and / or has high molecular weight risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. Myelofibrosis may be primary myelofibrosis, myelofibrosis that develops after polycythemia vera (post-PV MF), or myelofibrosis that develops 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 imeterstat, administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 administration cycles, each cycle comprising intravenous administration of approximately 7–10 mg / kg of imeterstat once every 3 weeks, intravenous administration of approximately 7–10 mg / kg of imeterstat once a week for 3 weeks, intravenous administration of approximately 2.5–10 mg / kg of imeterstat once every 3 weeks, or intravenous administration of approximately 0.5–9.4 mg / kg of imeterstat once every 3 weeks. In one particular embodiment, each administration cycle comprises intravenous administration of approximately 7–10 mg / kg of imeterstat once every 3 weeks. In another embodiment, each administration cycle comprises intravenous administration of approximately 9.4 mg / kg of imeterstat once every 3 weeks.

[0022] In some embodiments of methods for identifying or selecting patients 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 methods for identifying or selecting patients 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 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, 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, 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 mean relative telomere length of target cells present in a biological sample derived from an individual, determined to be less than or equal to the 50th percentile of the relative telomere length range determined from one or more known criteria, (b) Triple-negative status based on the absence of mutations in JAK2, CALR, and MPL, (c) A method is provided comprising administering a telomerase inhibitor to a patient who has one or more high molecular weight 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] This disclosure provides a method for identifying subjects with myelofibrosis (MF) for treatment with telomerase inhibitors, the method comprising measuring the hTERT expression level in a biological sample obtained from a 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 who are likely to benefit from treatment with the telomerase inhibitor.

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

[0028] This disclosure provides a method for monitoring therapeutic efficacy in subjects with myelofibrosis (MF), the method comprising measuring the hTERT expression level in a biological sample obtained from a patient after administration of a 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 of 50% or more in the hTERT expression level in the biological sample identifies subjects likely to benefit from treatment with a telomerase inhibitor. In certain embodiments, the telomerase inhibitor is imeterstat or a pharmaceutically acceptable salt thereof. In other embodiments, imeterstat is imeterstat sodium.

[0029] This disclosure provides a method for selecting patients most likely to benefit from treatment with a telomerase inhibitor, comprising: examining a patient for mean relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present in a patient-derived biological sample; and selecting a 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 within or equal to 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.

[0030] This disclosure provides a method for identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: obtaining a biological sample from a patient; determining the average relative length by analyzing the relative lengths of telomere nucleic acids in target cells present in the patient-derived biological sample; and identifying a patient if the patient has an average relative telomere length in target cells present in the patient-derived biological sample that is determined to be within or equal to the 50th percentile of a relative telomere length range determined from one or more known criteria, wherein the identified patient is most likely to benefit from treatment with a telomerase inhibitor.

[0031] This disclosure provides a method for treating a patient with myelofibrosis with a telomerase inhibitor, comprising administering the telomerase inhibitor to the patient if such a patient has an average relative telomere length in target cells present in a patient-derived biological sample, which is determined to be within 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] This disclosure provides a method for monitoring therapeutic efficacy in subjects with myelofibrosis (MF), the method comprising measuring the hTERT expression level in a biological sample obtained from a patient after administration of a 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 of 50% or more in the hTERT expression level in the biological sample identifies subjects 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 imeterstat or a pharmaceutically acceptable salt thereof. In other embodiments, imeterstat is imeterstat sodium.

[0033] This disclosure provides a method for identifying patients with myelofibrosis (MF) for treatment with telomerase inhibitors, 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 with the baseline hTERT expression level before administration of the telomerase inhibitor, thereby identifying patients in whom a decrease in the hTERT expression level in the biological sample is likely to benefit from treatment with the telomerase inhibitor.

[0034] This disclosure provides a method for monitoring therapeutic efficacy in subjects with myelofibrosis (MF), the method comprising measuring the telomerase activity level in a biological sample obtained from a patient after administration of a telomerase inhibitor, and comparing the telomerase activity level in the biological sample to 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 subjects likely to benefit from treatment with a 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 for selecting patients who are most likely to benefit from telomerase inhibitor treatment, (a) Testing a patient for triple-negative status, wherein the triple-negative status includes the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes, and / or (b) Testing 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 additional sex comb-like 1 (ASXL1), zest homolog 2 enhancer (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2), and / or (c) Examining a patient for average relative telomere length, by analyzing the relative length of telomere nucleic acids of target cells present in a biological sample derived from the patient, The aforementioned patient, If the aforementioned patient has triple-negative status, or If the aforementioned patient has HMR, or The selection includes the case where the patient has an average relative telomere length of 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, The selected patients are most likely to benefit from treatment with telomerase inhibitors. (Item 2) The method according to item 1, wherein the method comprises testing a patient for triple-negative status and selecting a patient if the patient does not have mutations in any of the JAK2, CALR, and MPL genes. (Item 3) The method according to item 1, wherein the method comprises examining the patient to determine whether the patient has HMR, and the selection of the patient includes 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 the method involves examining a patient for mean relative telomere length by analyzing the relative length of telomere 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 within or 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 that develops after polycythemia vera (post-PV MF), and myelofibrosis that develops after essential thrombocythemia (post-ET MF). (Item 6) The method described in any one of items 1 to 5, wherein the patient has not previously received JAK inhibitor therapy. (Item 7) The aforementioned patient, The patient had previously received JAK inhibitor therapy and was resistant to JAK inhibitor therapy. Have you previously received JAK inhibitor therapy and have experienced a relapse, or The method described in any one of items 1-5, for a person who has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. (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 includes bone marrow, peripheral blood, or a combination thereof. (Item 10) The use of telomerase inhibitors in the treatment of patients with myelofibrosis, (a) The patient is determined to have triple-negative status, which includes the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (b) The patient is determined to have high molecular weight risk (HMR), and 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 / or (c) Use of a patient having myelofibrosis in which cells present in a biological sample derived from the patient are determined to have an average relative telomere length that is less than or equal to the 50th percentile of a relative telomere length range determined from one or more known criteria. (Item 11) The use described in item 10, wherein the patient is determined to have triple-negative status, and the triple-negative status includes the absence of mutations in each of the JAK2, CALR, and MPL genes. (Item 12) The use described in item 10, wherein the patient is determined to have high molecular weight risk (HMR), and 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. (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 are determined to have an average relative telomere length that is less than or equal to the 50th percentile of the relative telomere length range determined from one or more known criteria. (Item 14) The use described in any one of items 10 to 13, wherein the myelofibrosis is selected from the group consisting of primary myelofibrosis, myelofibrosis that develops after polycythemia vera (post-PV MF), and myelofibrosis that develops after essential thrombocythemia (post-ET MF). (Item 15) The use described in any one of items 10-14, provided that the patient has not previously received JAK inhibitor therapy. (Item 16) The aforementioned patient, The patient had previously received JAK inhibitor therapy and was resistant to JAK inhibitor therapy. Have you previously received JAK inhibitor therapy and have experienced a relapse, or The user has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance, or uses as described in any one of items 10-14. (Item 17) The method according to any one of items 10 to 16, wherein the telomerase inhibitor is imetelstat. [Brief explanation of the drawing]

[0035] The above summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying figures. For the purpose of illustrating 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 fixtures shown.

[0036] [Figure 1]The waterfall plots of spleen volume reduction (SVR) at week 24 for the 4.7 mg / kg and 9.4 mg / kg treatment groups in Example 1 are shown. SVR is shown as the percentage change from baseline. [Figure 2] The waterfall plots show the reduction in total symptom score (TSS) at week 24 for the 4.7 mg / kg and 9.4 mg / kg treatment groups in Example 1. TSS is shown as a percentage change from baseline. [Figure 3] Kaplan-Meier plots of overall survival grouped by JAK2 / MPL / CALR gene mutation status: showing TN vs. non-TN (MUT) in the 4.7 mg / kg group. Specifically, Figure 3 shows the survival probabilities as a function of time for patients with triple-negative status (TN) and patients with at least one mutation (MUT). [Figure 4] Kaplan-Meier plots of overall survival grouped by JAK2 / MPL / CALR gene mutation status: showing TN vs. non-TN (MUT) in the 9.4 mg / kg group. Specifically, Figure 4 shows the survival probability as a function of time for patients with triple-negative status (TN) and patients with at least one mutation (MUT) in the 9.4 mg / kg group. [Figure 5] The Kaplan-Meier plots of overall survival as a function of time are shown, grouped according to patients in the 9.4 mg / kg vs. 4.7 mg / kg groups. [Figure 6] Kaplan-Meier plots of overall survival (OS) grouped by JAK2 / MPL / CALR gene mutation status: showing TN vs. non-TN in the 9.4 mg / kg group. Specifically, Figure 6 shows the survival probability as a function of time for patients with triple-negative status (TN) and patients with at least one mutation (non-TN) in the 9.4 mg / kg group. [Figure 7]Kaplan-Meier plots of overall survival (OS) grouped by JAK2 / MPL / CALR gene mutation status: showing TN vs. non-TN in the 4.7 mg / kg group. Specifically, Figure 7 shows the survival probability as a function of time for patients with triple-negative status (TN) and patients with at least one mutation (non-TN) in the 4.7 mg / kg group. [Modes for carrying out the invention]

[0037] This application is based on the finding that patients with myelofibrosis who are triple-negative (i.e., without mutations in each of JAK2, CALR, and MPL) and / or in the high molecular weight risk (HMR) category based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, may benefit from treatment with telomerase inhibitors such as imetelstat or imetelstat sodium. Patients with mutations in the ASXL1, EZH1, IDH1 / 2, and SRSF2 genes have an increased risk of premature death or leukemic transformation. These patients typically do not benefit from treatment with conventional therapies such as JAK inhibitors. Gisslinger et al., Blood, 128:1931 (2016). Therefore, the fact that these patients may benefit from treatment with telomerase inhibitors is unexpected and surprising.

[0038] Accordingly, this application provides a method for identifying patients most likely to benefit from treatment with a telomerase inhibitor such as imetelstat. The method includes examining or identifying patients to determine whether they have triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. This application also provides a method for treating myelofibrosis with a telomerase inhibitor such as imetelstat, the method including identifying patients who have triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or high molecular risk (HMR) based on the presence of mutations 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 patients. For clarity in the disclosure, and without limitation, the detailed description of the present invention is divided into subsections that describe or illustrate specific features, embodiments, or uses of the invention.

[0039] A.Definition As used herein, mutations in additional sex comb-like 1 (ASXL1), zest homolog 2 enhancer (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2) include any mutations in these genes that affect survival and disease progression in patients with myelofibrosis. Furthermore, as used herein, IDH1 / 2 includes IDH1 and IDH2. Exemplary mutations may be found in the following publications, each disclosure of which is incorporated for the purpose of disclosing 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 weight 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 with the nucleic acid sequence of SEQ ID NO: 5, for example, the EZH2 gene with the nucleic acid sequence of SEQ ID NO: 6, for example, the SRSF2 gene with the nucleic acid sequence of SEQ ID NO: 7, for example, the IDH1 gene with the nucleic acid sequence of SEQ ID NO: 8, the IDH2 gene with the nucleic acid sequence of SEQ ID NO: 9, and combinations thereof.

[0040] In some embodiments, the target mutations in the ASXL1 gene include 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, Examples of mutations include 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 include Q575X, Q588X, Y591X, Y591N, Q592X, S604F, L614F, Q623X, A627G, E635R, T638V, A640G, G646W, G658X, R678K, C687R, C687V, D690G, R693X, Y700X, G704R, G704W, E705X, Q708X, G710E, L721C, E727X, V751L, P763R, Q780X, and W796X. The mutations are 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 desired mutations in the EZH2 gene include the mutations 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 the 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 desired mutation in the SRSF2 gene includes a P95 mutation. In some embodiments, the mutation is a P95H mutation, a P95L mutation, or a P95R mutation.

[0043] In some embodiments, the desired mutation in the IDH1 / 2 gene includes the R132 and / or R140 mutation. In some embodiments, the mutation is the R132G mutation, the R132H mutation, or the R140Q mutation.

[0044] In a particular embodiment, the desired mutations include those listed below: [Table A]

[0045] As used herein, “triple-negative status,” “triple-negative,” or “TN” refers to a patient who is free from mutations in any of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. Triple-negative status can be determined based on the absence of mutations in, for example, the JAK2 gene having the nucleic acid sequence of SEQ ID NO: 2, the CALR gene having the nucleic acid sequence of, for example, SEQ ID NO: 3, and the MPL gene having the nucleic acid sequence of, for example, SEQ ID NO: 4.

[0046] In certain embodiments, triple-negative status includes the absence of mutations in the JAK2 gene, such as G335, F556, G571, V617, and / or V625. For example, triple-negative status may include the absence of the G335D mutation, F556V mutation, G571S mutation, V617F mutation, and / or V625S mutation in the JAK2 gene.

[0047] In certain embodiments, triple-negative status includes the absence of mutations in the MPL gene, such as T119, S204, P222, E230, V285, R321, S505, W515, Y591, and / or R592. For example, triple-negative status may include the absence of the 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, triple-negative status includes the absence of mutations in the CALR gene, such as mutations in L367, K368, E381, K385, and / or E396. For example, triple-negative status may include the absence of L367T mutations, K368R mutations, K385N mutations, E381A mutations, and / or E396del mutations in the CALR gene.

[0049] In a particular embodiment, the desired mutations include those listed below: [Table B]

[0050] As used herein, a patient has “failed” JAK inhibitor therapy when the disease was resistant, or when the patient was resistant to treatment, or when the patient was initially responsive to treatment but the disease relapsed.

[0051] As used herein, when referring to measurable values ​​such as quantity, duration, etc., the term “about” means to include a variation 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, as is appropriate for carrying out the disclosed method.

[0052] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to patients, such as mammals (a salt having a counterion that is mammalian safe for a given drug 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, which can be derived from a variety of organic and inorganic counterions known in the art, including, but only by example, sodium, and, if the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochlorides. Examples of pharmaceutically acceptable salts 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 salts” refers to compounds formed when the protons of an acid are replaced by cations such as metal cations or organic cations. Preferably, the salts are pharmaceutically acceptable salts. Examples of salts of the compound include those in which the compound is protonated by an inorganic or organic acid to form a cation, using an inorganic or organic acid conjugate base as the anionic component of the salt. Examples of salts of interest include, but are not limited to, salts of aluminum, ammonium, arginine, barium, benzathine, calcium, cesium, corinate, 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. For any oligonucleotide structures shown herein that include an internucleoside bond backbone, it should be understood that such oligonucleotides may include any convenient salt form. In some embodiments, the acidic form of the nucleoside bond is shown for simplicity. In some cases, the salt of the subject compound is a monovalent cationic salt. In certain cases, the salt of the subject compound is a divalent cationic salt. In some cases, the salt of the subject compound is a trivalent cationic salt. A “solvate” refers to a complex formed by a combination of solvent molecules and solute molecules or ions. 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 "stereoisomers" refer to compounds that have the same atomic bonding but different atomic arrangements in space. Examples of stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers. It should be understood that any group disclosed herein containing one or more substituents does not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically impossible. All stereoisomers are intended to be included within the scope of this disclosure.

[0055] Those skilled in the art will understand that other tautomer configurations of the groups described herein are possible. It should be understood that all tautomer forms of the subject compound are encompassed by the described structure, even if not specifically indicated, by one possible tautomer configuration of the groups of the compound.

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

[0057] Before any particular embodiment is described in more detail, it should be understood that the present invention is not limited to the specific embodiments described and, therefore, may vary. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit the scope of the present invention, as it is limited only by the appended claims.

[0058] Where a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates, each intervening value is included within the Invention up to one-tenth of the lower limit, between the upper and lower limits of that range and any other stated or intervening value within that range. These upper and lower limits of smaller ranges may independently be included in smaller ranges and are also included within the Invention, subject to any specifically excluded limitations within the stated range. If a stated range includes one or both of the limitations, the range excluding one or both of those included limitations is also included within the Invention.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein may also be used in carrying out or testing the present invention, but representative exemplary methods and materials are described herein.

[0060] All publications and patents cited herein are incorporated herein by reference as specifically and individually indicated, to disclose and describe methods and / or materials in relation to the cited publications. The citation of publications is for their prior disclosure prior to the filing date and should not be construed as acknowledging that the present invention has no prior rights to such publications for the sake of prior art. Furthermore, the dates of the publications provided may differ from the actual publication dates and these may need to be verified individually.

[0061] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. It should also be noted that claims may be constructed to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “simply,” “only,” or “negative” limitations relating to the enumeration of elements of the claims.

[0062] Each of the individual embodiments described and explained herein has distinct components and features, which can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events, or in any other logically possible order.

[0063] B. Identifying patients most likely to benefit from telomerase inhibitor therapy. In one embodiment, the disclosure provides a method for identifying or selecting myelofibrosis patients most likely to benefit from treatment with telomerase inhibitors. The method relies on identifying patients with triple-negative status (patients without mutations in any of the JAK2, CALR, and MPL genes) or patients at high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. These triple-negative or HMR patients are most likely to benefit from treatment with telomerase inhibitors such as imeterstat or imeterstat sodium.

[0064] Myelofibrosis may be primary myelofibrosis, myelofibrosis following polycythemia vera (post-PV MF), or myelofibrosis following essential thrombocythemia (post-ET MF). In certain embodiments, the patient has never 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 relapsed). 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 even more alternative embodiments, the patient has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy.

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

[0066] In one embodiment, the present invention provides a method for selecting patients most likely to benefit from telomerase inhibitor therapy by testing for one or more triple-negative statuses 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 weight 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 provides a method for selecting patients most likely to benefit from telomerase inhibitor therapy, by 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 for high molecular weight 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 provides a method for 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 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 most likely to benefit from treatment with a telomerase inhibitor.

[0067] Another embodiment of the present invention is a method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor, a. The patient shall be examined 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. Test for high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. b. When the patient has the following: i. Triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. Selecting patients who have high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, The selected patients are most likely to benefit from treatment with telomerase inhibitors.

[0068] A further embodiment of the present invention is a method for identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: testing patients for triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and selecting patients if they have triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, wherein the selected patients are most likely to benefit from treatment with a telomerase inhibitor. In one embodiment, the method also comprises: testing patients for high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and selecting patients 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 any other embodiment of these methods, high molecular weight risk (HMR) is determined by the presence of a mutation in at least one coding region (exon) of the ASXL1, EZH2, SRSF2, and IDH1 / 2 genes.

[0071] In certain embodiments, high molecular weight 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 mutations in ASXL1. In some embodiments, the method includes detecting the presence of mutations in EZH2. In some embodiments, the method includes detecting the presence of mutations in SRSF2. In some embodiments, the method includes detecting the presence of mutations 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 present invention, the present invention provides a method for identifying or selecting patients in a patient population who are most likely to benefit from treatment with telomerase inhibitors. In this method, the patient population is screened for patients with mutations in each of the JAK2, CALR, and MPL genes to identify triple-negative patients in the population. In an alternative embodiment, the method relies on identifying triple-negative patients who lack canonical mutations in each of the JAK2, MPL, and CALR genes.

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

[0073] Patient DNA samples are tested for the presence of mutations in each of the JAK2, CALR, and MPL genes using conventional techniques. Alternatively, patient DNA samples are tested for the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2 using conventional techniques. In certain embodiments, patient DNA samples are tested for (i) the presence 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 DNA sample testing may be a next-generation sequencing assay using the Illumina MiSeq platform, as described in Patel et al., Blood; 126(6): 790-797 (2015), and the disclosure relating to the DNA sample testing is incorporated herein.

[0075] C. Pharmacodynamics (PD) This disclosure is based in part on pharmacodynamic effects demonstrating a correlation between the response to telomerase inhibitor therapy in subjects with myelofibrosis and a reduction in telomerase hTERT expression levels from baseline. In some cases, a higher proportion of subjects who achieved a clinical response (splenic or symptomatic) to telomerase inhibitor therapy at week 24 achieved a reduction of 50% or more in hTERT RNA expression levels compared to subjects who did not achieve a response.

[0076] This disclosure provides a method for stratifying and selecting patients who are likely to benefit from telomerase inhibitor therapy for myelofibrosis and for monitoring response, relapse, and prognosis in those being treated.

[0077] Aspects of this disclosure include methods for selecting subjects having myelofibrosis (MF) for treatment with telomerase inhibitors, and methods for treating MF. Methods for monitoring therapeutic efficacy in subjects having MF are also provided. In some cases, the pharmacodynamic effect based on embodiments of the method for subjects is a reduction of 50% or more, e.g., 60% or more, 70% or more, 80% or more, or 90% or more of hTERT RNA expression.

[0078] Telomerase ribonucleic acid proteins consist of components or subunits, two of which are telomerase RNA template (hTR) and telomerase reverse transcriptase protein (hTERT). hTERT expression levels can be evaluated, determined, and / or measured using any convenient method. Various methods can be applied to amplify, detect, and measure mRNA of telomerase components or related proteins in body fluids. Target methods and assays that may be adapted for use in the methods in question include, but are not limited to, the methods 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] hTERT expression levels can be evaluated or measured in any convenient target cells or biological samples. Target cells may be any convenient cells from the patient, including, but not limited to, cells from the patient's bone marrow or peripheral blood. In some cases, target cells are isolated from the patient's bone marrow sample. In other cases, target cells are isolated from the patient's peripheral blood sample. Target cells may be granulocytes.

[0080] hTERT RNA expression levels 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 this disclosure relate to triple-negative status and / or the following genes, based on the absence of any mutations in the JAK2, CALR, and MPL genes (i.e., these genes are either free of mutations or lack mutations): The present invention includes a method for treating myelofibrosis in a subject (i.e., a patient) requiring treatment for myelofibrosis who has high molecular weight risk (HMR) based on the presence of a mutation in at least one of ASXL1, EZH2, SRSF2, and IDH1 / 2. One embodiment of the present invention is a method for treating myelofibrosis in a subject (i.e., a patient) requiring treatment for myelofibrosis who has triple-negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes (i.e., no mutations in these genes or lacking mutations). In one embodiment, myelofibrosis is primary myelofibrosis. In another embodiment, myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF). In an alternative embodiment, myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET 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 has “failed” the therapy (i.e., the disease was resistant, or the patient was resistant to the therapy, or initially responded to the therapy but the disease relapsed). In alternative embodiments of the treatment method, the patient has previously received JAK inhibitor therapy and has discontinued the 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 equivalents thereof.

[0083] The subjects are mammals requiring cancer treatment. Generally, the subjects are human patients. In some embodiments of the present invention, the subjects may be non-human mammals such as non-human primates, animal models (e.g., animals such as rats used for drug screening, characterization, and evaluation), 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 to obtain beneficial or desired outcomes, including clinical outcomes. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, reduction or improvement of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; a stabilization (i.e., non-exacerbating) state of the disease; prevention of disease spread; delay or slowing of disease progression; improvement or mitigation of the disease state; and remission (whether partial or total), whether detectable or undetectable. “Treatment” may also mean an extension of survival compared to the survival expected without 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 favorable telomerase inhibitor. Furthermore, any favorable telomerase inhibitor can be found for use in the therapeutic methods of interest. In some embodiments, the telomerase inhibitor is an oligonucleotide having telomerase inhibitory activity, particularly as defined in WO2005 / 023994 and / or WO2014 / 088785, the disclosures of which are incorporated herein by reference in their entirety. In some cases, one or more telomerase inhibitors (e.g., two or three telomerase inhibitors) can be administered to mammals to treat hematological malignancies.

[0086] Imetelstat In certain embodiments, the telomerase inhibitor is imeterstat, comprising its tautomers and salts thereof, e.g., pharmaceutically acceptable salts. Imeterstat 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): [ka] In the formula, "nps" represents the thiophosphoramide bond -NH-P(=O)(SH)-O- which links the 3'-carbon of one nucleoside to the 5'-carbon of an adjacent nucleoside.

[0087] In certain cases, the telomerase inhibitor is imetelstat sodium, including its tautomer. Imetelstat sodium is the sodium salt of imetelstat, which is a synthetic lipid-conjugated 13-mer oligonucleotide N3'→P5'-thio-phosphorumamide. Imetelstat sodium is a telomerase inhibitor that is a covalently lipid-conjugated 13-mer oligonucleotide (shown below) complementary to the human telomerase RNA (hTR) template region. The chemical name of imetelstat sodium is DNA, d(3'-amino-3'-deoxy-P-thio)(TAGGGTTAGACAA), 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 therefore does not have the side effects commonly observed with such therapies. [ka]

[0088] Unless otherwise indicated or the context makes clear, references to imeterstat herein also include its tautomers and salts, e.g., pharmaceutically acceptable salts. As stated above, imeterstat sodium is, in particular, the sodium salt of imeterstat. Unless otherwise indicated or the context makes clear, references to imeterstat sodium herein also include all of its tautomers.

[0089] Imeterstat and sodium imeterstat can be manufactured, formulated, or made available as described elsewhere (see, for example, 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 made clear from the context, references to imeterstat herein also include its salts. As stated above, sodium imeterstat is, in particular, the sodium salt of imeterstat.

[0090] Imetelstat targets the telomerase RNA template and inhibits telomerase activity and cell proliferation in various cancer cell lines and tumor xenografts in mice. Phase 1 trials in patients with breast cancer, non-small cell lung cancer and other solid tumors, multiple myeloma, or chronic lymphocytic leukemia have provided information on the drug's pharmacokinetics and pharmacodynamics. Subsequent Phase 2 trials in patients with essential thrombocythemia demonstrated thrombocytopenic activity associated with a significant reduction in JAK2 V617F and CALR mutant allele loadings. Imetelstat sodium is routinely administered intravenously. Other routes of administration, such as subarachnoid administration, intratumoral injection, and oral administration, are intended to be used in the implementation of the method in question. Imetelstat sodium can be administered at doses equivalent to those routinely used clinically. In certain embodiments, imetelstat sodium is administered as described elsewhere in this specification.

[0091] A particular embodiment follows any one of the other embodiments, wherein the imeterstat is limited to sodium imeterstat.

[0092] F. Pharmaceutical Compositions To facilitate administration, telomerase inhibitors (e.g., those described herein) can be formulated into various pharmaceutically acceptable forms for administration purposes. In some cases, telomerase inhibitors are administered as pharmaceutically acceptable compositions. The carrier or diluent of the pharmaceutically acceptable composition must be compatible with the other components of the composition and “acceptable” in the sense that it is not harmful to the recipient. The pharmaceutically acceptable composition may be a unitary dosage form particularly suitable for administration by oral, rectal, transdermal, parenteral injection, or inhalation. In some cases, administration may be carried out by intravenous injection. For example, when preparing a composition in oral dosage form, any of the usual pharmaceutically acceptable media such as water, glycol, oil, or alcohol may be used 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, and disintegrants may be used in the case of powders, pills, capsules, and tablets. Tablets and capsules are the most advantageous oral dosage unit forms because they are easy to administer, in which case solid pharmaceutically acceptable carriers are obviously used. For parenteral compositions, the carrier usually contains at least a large portion of sterile water, but may also contain other components, for example, to aid solubility. For example, an injectable solution may be prepared in which the carrier contains physiological saline, glucose solution, or a mixture of physiological saline and glucose solution. For example, an injectable solution may be prepared in which the carrier contains physiological saline, glucose solution, or a mixture of physiological saline and glucose solution. The injectable solutions containing the telomerase inhibitor described herein may be formulated in oil for long-acting effects. Suitable oils for this purpose include, 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 may also be prepared, in which case a suitable liquid carrier, suspension agent, etc., may be used. Solid preparations intended to be converted to liquid preparations immediately before use are also included.In compositions suitable for transdermal administration, the carrier optionally contains a penetration enhancer and / or a suitable wetting agent, and optionally combines in small amounts with suitable additives of any properties, which do not cause significant adverse effects on the skin. These additives may facilitate administration to the skin and / or may be useful in preparing the desired composition. The composition may be administered in various ways, for example, as a transdermal patch, a spot-on, or an ointment.

[0093] It is particularly advantageous to formulate the aforementioned pharmaceutical compositions into unit dosage forms for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically distinct unit suitable as a single dose, each unit containing a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. Examples of such unit dosage forms include tablets (including split or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions, and their separated multiple doses.

[0094] To enhance the solubility and / or stability of the drugs described herein in pharmaceutical compositions, it may be advantageous to employ α-, β-, or γ-cyclodextrins or their derivatives, particularly hydroxyalkyl-substituted cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Furthermore, cosolvents such as alcohols may improve the solubility and / or stability of telomerase inhibitors in pharmaceutical compositions.

[0095] Depending on the mode of administration, the pharmaceutical composition preferably comprises 0.05 to 99% by weight, more preferably 0.1 to 70% by weight, and 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, and even more preferably 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all proportions based on the total weight of the composition.

[0096] G. Dosage and Dosage Regimen The frequency of administration may be any frequency that reduces the severity of myelofibrosis symptoms without causing significant toxicity to the subject. For example, the frequency of administration may be approximately once every two months to approximately once a week, alternatively once every month to approximately twice a month, alternatively once every six weeks, approximately once every five weeks, alternatively once every four weeks, alternatively once every three weeks, alternatively once every two weeks, or alternatively once every week. The frequency of administration may remain constant or may change during the course of treatment. The course of treatment with a composition containing one or more telomerase inhibitors may include rest periods. For example, a composition containing telomerase inhibitors may be administered weekly for three weeks, followed by a two-week rest period, and such a regimen may be repeated multiple times. As with the effective dose, various factors may influence the actual frequency of administration used for a particular application. For example, the effective dose, duration of treatment, use of multiple therapeutic agents, route of administration, and the severity of myelofibrosis and related symptoms may necessitate an increase or decrease in the frequency of administration.

[0097] The effective period for administering a composition containing a telomerase inhibitor (e.g., imeterstat or imeterstat sodium) may be any period that reduces the severity of myelofibrosis symptoms (as described herein) without causing significant toxicity to the subject. Therefore, the effective period may vary from one month to several months or several years (e.g., one month to two years, one month to one year, three months to two years, three months to ten months, or three months to eighteen months). Generally, the effective period for treating myelofibrosis may range from two months to twenty months. In some cases, the effective period may be as long as the individual subject is alive. Several factors may influence the actual effective period used for a particular treatment. For example, the effective period may vary depending on the frequency of administration, the effective dose, the use of multiple therapeutic agents, the route of administration, and the severity of myelofibrosis and associated 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 myelofibrosis symptoms is decreasing. For example, the severity of myelofibrosis symptoms (as described herein, for example) can be assessed using biopsy techniques.

[0099] The telomerase inhibitors used in the methods described herein may be administered at any therapeutically effective dose, including doses equivalent to those routinely used clinically. Specific dose regimens (e.g., recommended effective doses) for known and approved anticancer agents are known to physicians and are described in product descriptions, for example, in *Physicians' Desk Reference, 2003, 57th Ed., Medical Economics Company, Inc., Oradell, NJ* and *Goodman & Gilman's The Pharmaceutical 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 dose of the telomerase inhibitor, imeterstat sodium, administered to the subject is approximately 1.0 mg / kg to approximately 13.0 mg / kg. In other embodiments, the dose of the telomerase inhibitor is approximately 4.5 mg / kg to approximately 11.7 mg / kg, or approximately 6.0 mg / kg to approximately 11.7 mg / kg, or approximately 6.5 mg / kg to approximately 11.7 mg / kg. In some embodiments, the dose of the telomerase inhibitor is at least approximately 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 g, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2m g / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 10.1mg / kg, 10.2mg / kg , 10.3mg / kg, 10.4mg / kg, 10.5mg / kg, 10.6mg / kg, 10.7mg / kg, 10.8mg / kg, 10.9mg / kg, 11mg / kg, 11.1mg / kg, 11.2mg Contains one of the following: / kg, 11.3mg / kg, 11.4mg / kg, 11.5mg / kg, 11.6mg / kg, 11.7mg / kg, 11.8mg / kg, 11.9mg / kg, 12mg / kg, 12.1mg / kg, 12.2mg / kg, 12.3mg / kg, 12.4mg / kg, 12.5mg / kg, 12.6mg / kg, 12.7mg / kg, 12.8mg / kg, 12.9mg / kg, or 13mg / kg.

[0101] In some embodiments, the effective dose of the telomerase inhibitor administered to an individual includes at least one of the following: approximately 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 dose of the telomerase inhibitor administered to an individual is approximately 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 dose of telomerase inhibitor administered to an individual is less than any of the following amounts: approximately 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 for pharmaceutical compositions containing telomerase inhibitors include, but are not limited to, daily, every other day, twice a week, three times a week, weekly, weekly, three weeks out of four, once every three weeks, once every two weeks, and weekly for two weeks out of three. In some embodiments, the pharmaceutical composition is administered approximately once a 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, four, five, six, or seven times a week (i.e., daily), or three times a day or twice a day. In some embodiments, the interval between doses is shorter than approximately 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between doses is longer than approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there are no breaks in the medication schedule. In some embodiments, the interval between doses is approximately 1 week or less.

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

[0104] In certain embodiments of this method, imeterstat is administered over one, two, three, four, five, six, seven, eight, or more than eight administration cycles, each cycle comprising intravenous administration of approximately 7–10 mg / kg of imeterstat once every three weeks, intravenous administration of approximately 7–10 mg / kg of imeterstat once a week for three weeks, intravenous administration of approximately 2.5–10 mg / kg of imeterstat once every three weeks, or intravenous administration of approximately 0.5–9.4 mg / kg of imeterstat once every three weeks. In certain cases, each administration cycle comprises intravenous administration of approximately 7–10 mg / kg of imeterstat once every three weeks. In other cases, each administration cycle comprises intravenous administration of approximately 9.4 mg / kg of imeterstat once every three weeks.

[0105] In one embodiment, imeterstat is administered intravenously at a dose of approximately 7-10 mg / kg once every three weeks, following premedication with an antihistamine, a corticosteroid, or both. In another embodiment, imeterstat is administered intravenously at a dose of approximately 9.4 mg / kg, or alternatively, approximately 7.0 mg / kg to approximately 9.8 mg / kg, once every three weeks, following premedication with an antihistamine, a corticosteroid, or both.

[0106] In a particular embodiment, imetelstat is administered once every three weeks for at least three cycles at a dose of approximately 7.5 mg / kg, or alternatively, approximately 7.0 mg / kg to approximately 7.7 mg / kg, thereafter increasing the dose. In a particular embodiment, the dose of imetelstat is such that the ANC and the lowest platelet point are each approximately 1.5 × 10⁻⁶. 9 / L~Approx. 75×10 9 Provided that the dose has not decreased at / L and there are no non-hematological toxicities of grade ≥ 3, the dose may be increased to approximately 9.4 mg / kg, or approximately 8.8 mg / kg to approximately 9.6 kg / mg.

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

[0108] In certain embodiments, imetelstat may be administered via a regimen involving dose reduction. In one embodiment, the patient is initially given approximately 9.4 mg / kg every three weeks, then the dose is reduced to approximately 7.5 mg / kg every three weeks, and then to approximately 6.0 mg / kg every three weeks.

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

[0110] Aspects of the methods described herein include identifying or selecting patients most likely to benefit from treatment based on relative telomere length in the patient's target cells (e.g., as described herein). Target cells may be any favorable cells of the patient, including, but not limited to, cells from the patient's bone marrow or peripheral blood. In some cases, target cells are isolated from the patient's bone marrow sample. In other cases, target cells are isolated from the patient's peripheral blood sample. Target cells may 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 less than or equal to the median or mean telomere length compared to a preferred control, e.g., one or more known criteria described herein. Therefore, the method of the subject may further include determining relative telomere length by analyzing the relative length of telomere nucleic acids of target cells present in a biological sample derived from an individual, and selecting individuals who would benefit from treatment with a telomere enzyme inhibitor if the average relative telomere length in target cells present in a biological sample derived from an individual is determined to be below the 50th percentile of a relative telomere length range determined from one or more known criteria, for example, below the 45th percentile, 40th percentile, 35th percentile, 30th percentile, 25th percentile, 20th percentile, or less of a relative telomere length range determined from one or more known criteria.

[0111] In some examples of the method, one or more known criteria are the range of telomere lengths established from multiple naturally occurring target cells (e.g., as described herein) from multiple individuals diagnosed with the disease. In certain examples of the method, one or more known criteria are characterized cell lines. A “characterized cell line” means that the relative telomere 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 a biological sample is determined to be less than or equal to the median or mean telomere length. In some embodiments, the telomere length of cancer cells present in a biological sample is determined to be less than or equal to 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 target cells can be determined using any convenient assay, including but not limited to qPCR, telo-FISH, or Southern blot assays, as described by Bassett et al. in U.S. Patent No. 9,200,327. In one embodiment, telomere length can be determined by measuring the average length of terminal restriction fragments (TRFs). A TRF is defined as the length (usually the average length) of the fragment resulting from the complete digestion of genomic DNA with 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 frequently cleaves within the genomic DNA but not within the telomere sequence. In some cases, the restriction enzyme has four base recognition sequences (e.g., AluI, HinfI, RsaI, and Sau3A1) and is used alone or in combination. The resulting terminal restriction fragments contain both telomere repeats and subtelomere DNA. Subtelomere DNA is DNA sequences adjacent to tandem repeats of telomere sequences and contains telomere repeats scattered within variable telomere-like sequences. Digested DNA is separated by electrophoresis and blotted onto a support such as a membrane. Fragments containing telomere sequences are detected by hybridizing a probe, i.e., a labeled repeat sequence, into the membrane. Visualization of telomere-containing fragments allows for the calculation of the mean length of terminal restriction enzyme fragments (Harley, CBe 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 therefore shows the median and mean telomere lengths of all cells.

[0114] In another embodiment, 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). Flow cytometry is a variation of the FISH technique. When the starting material is tissue, the cell suspension is generally prepared by mechanical separation and / or treatment with proteases. The cells are fixed with a fixative and hybridized with a fluorescently labeled telomere sequence-specific probe, preferably a PNA probe. After hybridization, the cells are washed and then analyzed by FACS. After appropriately subtracting background fluorescence, the fluorescence signal is measured for cells in Go / G1. This technique is suitable for rapid estimation of telomere length for a large number of samples. Similar to TRF, telomere length is the average length of telomeres within a cell.

[0115] In other embodiments, the median or mean telomere length of cells in 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, causing the dye to fluoresce. 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-logarithmic scale. A threshold is determined for detecting fluorescence above the background. The cycle at which fluorescence from the sample exceeds the threshold is called the cycle threshold (Ct). The relative amount of DNA can be calculated because, theoretically, the amount of DNA doubles with each cycle during the logarithmic phase. The baseline is the initial cycle 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 with a fluorescent label, such as Cy-3, fluorescein, or rhodamine. The probe in this method is an oligonucleotide designed to specifically hybridize to the telomere sequence. Generally, the probe is at least 8 nucleotides long, e.g., 12-20 nucleotides or longer. In one embodiment, the probe is an oligonucleotide containing naturally occurring nucleotides. In another embodiment, the probe is a peptide nucleic acid, which has a higher Tm than similar natural sequences, allowing for the use of more stringent hybridization conditions. Cells may be treated with a drug such as colsemid to induce cell cycle arrest at metaphase, providing metaphase chromosomes for hybridization and analysis. In some embodiments, cellular DNA may also be stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI).

[0117] Digital images of intact metaphase chromosomes are obtained, and the fluorescence intensity of probes hybridized to telomeres is quantified. This allows for the measurement of telomere lengths of individual chromosomes, in addition to the mean or median telomere length within the cell, avoiding problems associated with the presence of subtelomere DNA (Zjilmans, J Met al., Proc. Natl. Acad Sci. USA 94:7423-7428 (1997), Blasco, MA et al. Cell 91:25-34 (1997), incorporated by reference). The intensity of the fluorescence signal correlates with telomere length, with brighter fluorescence signals indicating longer telomeres.

[0118] In a particular embodiment, the present invention relates to a telomerase inhibitor for use in a method for treating myelofibrosis, the method being: The goal is to identify patients who are most likely to benefit from treatment with telomerase inhibitors, and to select patients who are most likely to benefit from treatment with telomerase inhibitors. (a) Triple-negative status based on the absence of any mutations in the JAK2, CALR, and MPL genes. (b) High molecular weight 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 include, The presence of (a), (b), or (c) indicates that the patient is most likely to benefit from treatment with a telomerase inhibitor and from administering an effective amount of a telomerase inhibitor to the patient. In certain embodiments, the present invention relates to a telomerase inhibitor for use in a manner defined in any of the other embodiments.

[0119] A further embodiment of the present invention is a telomerase inhibitor for use in the treatment of myelofibrosis, wherein the use comprises (a) screening a patient to determine whether such a patient is triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or is at high molecular weight (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 the telomerase inhibitor to the patient if such a patient is triple-negative status based on the absence of mutations in each of JAK2, CALR, and MPL, and / or is at high molecular weight (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. A further embodiment of the present invention is a telomerase inhibitor for use in the treatment of myelofibrosis, wherein the use comprises (a) screening a patient to determine whether such a patient is triple-negative based on the absence of mutations in each of JAK2, CALR, and MPL, and (b) administering the patient a telomerase inhibitor if such a patient is triple-negative.

[0120] A further 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 triple-negative based on the absence of mutations in each of JAK2, CALR, and MPL, and / or is at high molecular weight (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 a patient if such a patient is triple-negative based on the absence of mutations in each of JAK2, CALR, and MPL, and / or is at high molecular weight (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. A further 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 triple-negative based on the absence of mutations in each of JAK2, CALR, and MPL, and (b) if such a patient is triple-negative, administering the patient a telomerase inhibitor.

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

[0122] In other embodiments of the present invention, high molecular weight risk (HMR) may 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 any combination thereof.

[0123] In other embodiments of the present invention, telomerase activity and hTERT expression levels in patient-derived biological samples can be determined to evaluate pharmacodynamic effects and / or monitor patients being treated with telomerase inhibitors. Telomerase activity can be measured using the TRAP (Telomere Repeat Sequence Amplification Protocol) telomerase activity assay. hTERT expression levels can be determined by measuring the hTERT RNA expression level in cells in the biological sample using Northern blotting or sequential gene expression analysis (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 present invention relates to the use of telomerase inhibitors for the treatment of myelofibrosis as defined in any of the other embodiments.

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

[0127] Clause 1. A method for identifying patients who are most likely to benefit from treatment with telomerase inhibitors, (a) Testing patients 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, (b) Select patients who have triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and the selected patients are most likely to benefit from treatment with telomerase inhibitors.

[0128] Clause 2. A method for identifying patients who are most likely to benefit from treatment with telomerase inhibitors, (c) Examining 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. Test for high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. (d) Selecting patients who have the following: i. Triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. Selecting patients with high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, The selected patients are most likely to benefit from treatment with telomerase inhibitors.

[0129] Clause 3. A method for identifying patients who are most likely to benefit from treatment with telomerase inhibitors, (e) Obtaining DNA samples from patients, (f) Test patient-derived DNA samples for triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, (g) Selecting patients if they have triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, The selected patients are most likely to benefit from treatment with telomerase inhibitors.

[0130] Clause 4. A method for identifying patients who are most likely to benefit from treatment with telomerase inhibitors, (h) Obtaining DNA samples from patients, (i) DNA samples derived from the patient, i. Triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. Test for high molecular weight risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. (j) The patient i. Triple-negative status based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. Selecting patients who have high molecular weight 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 most likely to benefit from treatment with telomerase inhibitors.

[0131] Clause 5. Use of telomerase inhibitors in the treatment of patients with myelofibrosis who are determined to have triple-negative status, The use of telomerase inhibitors, in which triple-negative status includes the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes.

[0132] Clause 6. Use of telomerase inhibitors in the treatment of patients with myelofibrosis who are determined to have high molecular weight risk (HMR), Having HMR is the use of telomerase inhibitors, which involves the presence of a mutation in at least one gene selected from the group consisting of additional sex comb-like 1 (ASXL1), zest homolog 2 enhancer (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0133] Clause 7. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein cells present in a patient-derived biological sample are determined to have an average relative telomere length that is less than or equal to the 50th percentile of a relative telomere length range determined from one or more known criteria.

[0134] Clause 8. Use of telomerase inhibitors in the manufacture of pharmaceuticals for the treatment of patients with myelofibrosis in which the patient is determined to have triple-negative status, wherein the triple-negative status includes the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes.

[0135] Clause 9. Use of telomerase inhibitors in the manufacture of pharmaceuticals for the treatment of patients with myelofibrosis in whom the patient is determined to have high molecular weight risk (HMR), wherein having HMR includes the presence of a mutation in at least one gene selected from the group consisting of additional combine 1 (ASXL1), zest homolog 2 enhancer (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0136] Clause 10. Use of telomerase inhibitors in the manufacture of agents for the treatment of patients with myelofibrosis, wherein cells present in a patient-derived biological sample are determined to have an average relative telomere length that is determined to be less than or equal to the 50th percentile of a range of relative telomere lengths determined from one or more known criteria.

[0137] The following examples are provided for illustrative purposes only, and are not limiting. [Examples]

[0138] Example 1: Imetelstat sodium is an effective treatment for patients with relapsed or Janus kinase (JAK) inhibitor-resistant moderate-grade 2 (int-2) or high-risk myelofibrosis (MF). 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 study of 33 patients with moderate-grade 2 (int-2) or high-risk myelofibrosis (MF), 48% of whom had previously been treated with Janus kinase inhibitors (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] method A randomized, multicenter, phase 2 trial of two doses of imetelstat sodium (9.4 mg / kg or 4.7 mg / kg IV every three weeks) was conducted in adults with a Dynamic International Prognostic Scoring (DIPSS) score of int-2 or high-risk MF who were relapsed / resistant to previous 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). Diagnosis of primary, essential thrombocythemia-associated, or polycythemia vera-associated MF was required. Other eligibility criteria included measurable splenomegaly (by magnetic resonance imaging [MRI]), active MF-related systemic symptoms, and platelet count ≥75 × 10⁶ 9 / L was included. The primary endpoints were splenic response rate (achieving ≥35% splenic volume reduction [SVR] by MRI at 24 weeks) and symptom response rate (achieving ≥50% reduction in total symptom score [TSS] according to the Myelofibrosis Symptom Assessment Form (MFSAF) v2 at 24 weeks). Secondary endpoints included safety, overall survival (OS), treatment response, molecular response, and pharmacokinetic and pharmacodynamic relationships.

[0140] result 107 patients were enrolled in 55 facilities (48 at 4.7 mg / kg and 59 at 9.4 mg / kg). Baseline characteristics are shown in Table 1 below. Additionally, the median time to JAKi was 23 months (0.9–89.7), and the median platelet count was 147 × 10⁶. 9 The result was / L. Triple-negative (TN, i.e., no JAK2, MPL, or CALR mutations) accounted for 24.8% of patients, and 67.6% were considered high-molecular-weight risk (HMR, i.e., ASXL1, EZH2, SRSF2, or IDH1 / 2 mutations ≥1). [Table 1]

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

[0142] At the clinical cutoff, patients were followed up for a median of 22.6 months (0.2–27.4 months), and the median treatment duration was 6.2 months (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 patients (10.2%) in the 9.4 mg / kg group had a splenic response on each MRI, while none in the 4.7 mg / kg group (see Figure 1). Nineteen patients (32%) in the 9.4 mg / kg group and three patients (6%) 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 was not reached in the 9.4 mg / kg group, but the median OS in the 4.7 mg / kg group was 19.9 months. The 18-month survival rates for the 9.4 mg / kg and 4.7 mg / kg groups were 76.7% and 62.9%, respectively. Sensitivity analysis yielded similar results by discontinuing patients at dose escalation for subsequent JAKi therapy or stem cell transplantation. In the 9.4 mg / kg group, an association was observed between TN patients and OS patients (median OS was not reached in TN patients, compared to 23.6 months in non-TN patients). The splenic 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%), while at 4.7 mg / kg, they 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 cytopenia resolved within 4 weeks. Grade 3 / 4 LFT elevation was observed in 7 patients during the study. No imetelstat-related hepatotoxicity was observed, as confirmed by an independent hepatic review committee.

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

[0146] Triple-negative vs. OS The subjects were grouped by their JAK2 / MPL / CALR gene mutation status: triple-negative (TN, no mutations in any of the JAK2 / MPL / CALR genes), and non-TN (mutation in one or more 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). However, in the 4.7 mg / kg group, the median OS with 95% confidence was 22.3 (17, NE) and 20.3 (18.3, NE) for TN subjects and non-TN subjects, respectively. In the 9.4 mg / kg group, the triple-negative (TN) group showed a lower mortality rate compared to the non-TN group (see Table 2, Figures 3 and 4). [Table 2]

[0147] At the second clinical cutoff, the triple-negative (TN) group showed a lower mortality rate 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. 24-week response 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 versus week 24 response In the 9.4 mg / kg group, a higher response rate (SVR or TSS) was observed in the low molecular weight (LMR) group or high molecular weight (HMR) group with only one mutation (mut) compared to the HMR group with more than one mutation (see Table 5). [Table 5]

[0150] For subjects receiving 9.4 mg / kg, the following factors were associated with clinical response or overall survival (OS). Triple Negative (TN): Response (SVR or TSS) was enhanced in TN subjects. Median OS was not estimable for TN, non-TN = 23.6 months, and Molecular risk: Response (SVR or TSS) was enhanced in subjects with HMR having only one mutation, and the response was observed in patients with HMR having more than one mutation treated with 9.4 mg / kg of imetelstat.

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

[0152] In the 9.4 mg / kg group, a better overall survival (OS) trend was observed in subjects with shorter baseline time thresholds (TLs), i.e., subjects whose baseline TL was below the median TL. [Table 6]

[0153] Baseline TL vs. Week 24 Response Baseline telomere length (TL): 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 exhibited a splenic response. 34.5% (10 / 29) of subjects with shorter baseline TL and 25% (6 / 24) of subjects with longer baseline TL exhibited a TSS response.

[0154] In the 9.4 mg / kg group, at week 24, subjects with shorter baseline timelines (TLs) showed enhanced high response rates (SVR or TSS) compared to subjects with longer TLs (Table 7). [Table 7]

[0155] Example 3 Dose-dependent pharmacodynamic (PD) effects The pharmacodynamic effects of imetelstat were evaluated by analyzing telomerase activity and hTERT. Among subjects with available baseline and post-treatment data, 23 subjects (51.1%) in the 9.4 mg / kg group and 10 subjects (29.4%) in the 4.7 mg / kg group achieved a >=50% reduction in telomerase activity from baseline, demonstrating a PD effect and correlating with antitumor activity from in vivo preclinical xenograft models. In addition, 35 subjects (61.4%) in the 9.4 mg / kg group and 20 subjects (47.7%) in the 4.7 mg / kg group achieved a >=50% reduction in hTERT RNA levels from baseline (Table 8). Therefore, a dose-dependent PD effect was demonstrated, indicating target engagement. [Table 8]

[0156] Example 4 Relationship between PD effect and response at week 24 A higher proportion of subjects with a splenic response (83.3%) achieved at least a >=50% reduction in hTERT RNA expression levels compared to subjects with a non-splenic response (55.6%), and a higher proportion of subjects with a TSS response achieved at least a >=50% reduction in hTERT RNA expression levels compared to non-TSS responders (Table 9). hTERT RNA expression levels were measured from whole blood samples collected from patients before and after treatment. [Table 9]

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

[0158] The aspects of the subject matter described herein, including embodiments, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the description, certain non-limiting aspects of this disclosure are provided below. As will be obvious to those skilled in the art by reading this disclosure, each individually numbered aspect may be used or combined with any preceding or succeeding individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects expressly provided below. 1. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, where the patient is determined to have triple-negative status, Triple negative status is used, including the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. 2. The use described in embodiment 1, wherein the myelofibrosis is primary myelofibrosis. 3. The use described in embodiment 2, wherein the myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF). 4. The use described in Embodiment 2, wherein the myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF). 5. Use as described in any one of embodiments 1 to 4, where the patient has not previously received JAK inhibitor therapy. 6. Use as described in any one of the descriptions in paragraphs 1 to 4, where the patient has previously received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 7. Use as described in any one of the descriptions in descriptions 1 to 4, where the patient has previously received JAK inhibitor therapy and is experiencing a relapse. 8. Use according to any one of embodiments 1 to 4, where the patient has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy 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 the imetelstat is imetelstat sodium. 11. The telomerase inhibitor is imetelstat, administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 administration cycles, each cycle being Administer approximately 7-10 mg / kg of imetelstat intravenously once every three weeks. Administer approximately 7-10 mg / kg of imetelstat intravenously once a week for 3 weeks. Administer approximately 2.5-10 mg / kg of imetelstat intravenously once every three weeks, or The use according to embodiment 10, which includes administering approximately 0.5 to 9.4 mg / kg of imetelstat intravenously once every three weeks. 12. The use according to embodiment 11, wherein each administration cycle comprises intravenous administration of approximately 7-10 mg / kg of imetelstat once every three weeks. 13. The use according to embodiment 12, wherein each administration cycle comprises intravenous administration of approximately 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 lengths of telomere nucleic acids in target cells present in a patient-derived biological sample. 15. 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 within or below the 50th percentile of the relative telomere length range determined from one or more known criteria. 16. Use according to any one of embodiments 1 to 15, further comprising screening a patient to determine whether the patient has high molecular weight 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 modifying the dosage, frequency of administration, or treatment course administered to the subject of the telomerase inhibitor. 20. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, where the patient is determined to be at high risk for high molecular weight (HMR), Having HMR involves the presence of a mutation in at least one gene selected from the group consisting of additional sex comb-like enzyme 1 (ASXL1), zest homolog 2 enhancer (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2). 21. The use described in embodiment 20, wherein the myelofibrosis is primary myelofibrosis. 22. Myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF), as described in Embodiment 21. 23. Myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF), as described in Embodiment 21. 24. Use according to any one of embodiments 20 to 23, wherein the patient has not previously received JAK inhibitor therapy. 25. Use according to any one of embodiments 20 to 23, wherein the patient has previously received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 26. Use as described in any one of descriptions 20 to 23, where the patient has previously received JAK inhibitor therapy and is experiencing a relapse. 27. Use according to any one of embodiments 20 to 23, where the patient has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 28. The use according to any one of embodiments 20 to 27, wherein the telomerase inhibitor is imetelstat. 29. The use according to embodiment 28, wherein the imetelstat is imetelstat sodium. 30. The telomerase inhibitor is imetelstat, administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, with each cycle being: Administer approximately 7-10 mg / kg of imetelstat intravenously once every three weeks. Administer approximately 7-10 mg / kg of imetelstat intravenously once a week for 3 weeks. Administer approximately 2.5-10 mg / kg of imetelstat intravenously once every three weeks, or The use according to embodiment 28, which includes administering approximately 0.5 to 9.4 mg / kg of imetelstat intravenously once every three weeks. 31. The use according to embodiment 30, wherein each administration cycle comprises intravenous administration of approximately 7-10 mg / kg of imetelstat once every three weeks. 32. The use according to embodiment 31, wherein each administration cycle comprises intravenous administration of approximately 9.4 mg / kg of imetelstat once every three weeks. 33. Use according to any one of embodiments 20 to 32, further comprising determining the average relative telomere length by analyzing the relative lengths of telomere nucleic acids in target cells present in a patient-derived biological sample. 34. The use according to any one of embodiments 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 within or below the 50th percentile of the relative telomere length range determined from one or more known criteria. 35. Use according to any one of embodiments 20 to 34, further comprising screening a patient to determine whether the patient has triple-negative status, wherein triple-negative status includes the absence of mutations in each of the genes selected from the group consisting of JAK2, CALR, and MPL. 36. The use according to any one of embodiments 20 to 35, further comprising evaluating the hTERT expression level in a biological sample obtained from a patient after administration of a telomerase inhibitor. 37. The use according to embodiment 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 embodiments 36 to 37, further comprising changing the dosage, frequency of administration, or treatment course administered to the subject of the telomerase inhibitor. 39. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein cells present in a patient-derived biological sample are determined to have an average relative telomere length that is less than or equal to the 50th percentile of the relative telomere length range determined from one or more known criteria. 40. The use described in embodiment 39, wherein the myelofibrosis is primary myelofibrosis. 41. Myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF), as described in embodiment 40. 42. Myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF), as described in embodiment 40. 43. Use according to any one of embodiments 39 to 42, wherein the patient has not previously received JAK inhibitor therapy. 44. Use according to any one of embodiments 39 to 42, wherein the patient has previously received JAK inhibitor therapy and the patient was resistant to JAK inhibitor therapy. 45. Use as described in any one of descriptions 39 to 42, where the patient has previously received JAK inhibitor therapy and is experiencing a relapse. 46. ​​Use according to any one of embodiments 39 to 42, where the patient has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 47. The use according to any one of embodiments 39 to 46, wherein the telomerase inhibitor is imetelstat. 48. The use according to embodiment 47, wherein the imetelstat is imetelstat sodium. 49. The telomerase inhibitor is imetelstat, administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, with each cycle being: Administer approximately 7-10 mg / kg of imetelstat intravenously once every three weeks. Administer approximately 7-10 mg / kg of imetelstat intravenously once a week for 3 weeks. Administer approximately 2.5-10 mg / kg of imetelstat intravenously once every three weeks, or The use according to embodiment 47, which includes administering approximately 0.5 to 9.4 mg / kg of imetelstat intravenously once every three weeks. 50. The use according to embodiment 49, wherein each administration cycle comprises intravenous administration of approximately 7-10 mg / kg of imetelstat once every three weeks. 51. The use according to embodiment 50, wherein each administration cycle comprises intravenous administration of approximately 9.4 mg / kg of imetelstat once every three weeks. 52. Use according to any one of embodiments 39 to 51, further comprising determining the average relative telomere length by analyzing the relative lengths of telomere nucleic acids in cells present in a patient-derived biological sample. 53. The use according to any one of embodiments 39 to 52, further comprising evaluating the hTERT expression level in a biological sample obtained from a patient after administration of a telomerase inhibitor. 54. The use according to embodiment 53, wherein the hTERT expression level is reduced by 50% or more compared to the baseline hTERT expression level before administration of the telomerase inhibitor. 55. The use according to any one of embodiments 53 to 54, further comprising changing the dosage, frequency of administration, or treatment course administered to the subject of the telomerase inhibitor. 56. A method for selecting patients who are most likely to benefit from treatment with telomerase inhibitors, This involves testing patients for triple-negative status, which includes testing for the absence of mutations in each of the JAK2, CALR, and MPL genes. This includes selecting patients who have triple-negative status, The selected patients are most likely to benefit from treatment with telomerase inhibitors. 57. The method according to embodiment 56, wherein the patient has myelofibrosis. 58. The method according to aspect 57, wherein the myelofibrosis is primary myelofibrosis. 59. The method according to aspect 57, wherein the myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF). 60. The method according to aspect 57, wherein the myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF). 61. The method according to any of embodiments 56 to 60, wherein the patient has not previously received JAK inhibitor therapy. 62. The patient, I have previously received JAK inhibitor therapy. Previously received JAK inhibitor therapy and failed JAK inhibitor therapy; or The method according to any one of embodiments 56 to 60, wherein the patient has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 63. The method according to any one of embodiments 56 to 60, wherein the patient has previously received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 64. The method according to any one of embodiments 56 to 60, wherein the patient has previously received JAK inhibitor therapy and has relapsed. 65. The method according to any one of embodiments 56 to 60, wherein the patient has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 66. The method according to any one of embodiments 56 to 65, further comprising administering a telomerase inhibitor to the patient. 67. The method according to embodiment 66, wherein the telomerase inhibitor is imetelstat. 68. The method according to embodiment 67, wherein the imetelstat is imetelstat sodium. 69. The method according to any one of embodiments 56 to 68, further comprising obtaining a sample containing DNA from a patient. 70. The method according to embodiment 69, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 71. The step of obtaining a sample from the patient, Obtaining bone marrow samples, peripheral blood samples, or combinations thereof, The method according to embodiment 70, comprising isolating DNA from a bone marrow sample, a peripheral blood sample, or a combination thereof. 72. The step of obtaining a sample from the patient, Obtaining bone marrow samples from patients, Isolating cells from bone marrow samples, The method according to embodiment 70, comprising extracting DNA from isolated cells. 73. The step of obtaining a sample from the patient, Obtaining peripheral blood samples from patients, Isolating cells from peripheral blood samples, The method according to embodiment 70, comprising extracting DNA from isolated cells. 74. A method for selecting patients who are most likely to benefit from treatment with telomerase inhibitors, The test involves examining a patient to determine whether or not they have 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. If a patient has HMR, this includes selecting the patient, The selected patients are most likely to benefit from treatment with telomerase inhibitors. 75. The method according to embodiment 74, wherein the patient has myelofibrosis. 76. The method according to aspect 75, wherein the myelofibrosis is primary myelofibrosis. 77. The method according to aspect 75, wherein the myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF). 78. The method according to aspect 75, wherein the myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF). 79. The method according to any of embodiments 74 to 78, wherein the patient has not previously received JAK inhibitor therapy. 80. The patient, I have previously received JAK inhibitor therapy. If you have previously received JAK inhibitor therapy and have experienced a failure with JAK inhibitor therapy, The method according to any one of embodiments 74 to 78, wherein the patient has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 81. The method according to any one of embodiments 74 to 78, wherein the patient has previously 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 previously received JAK inhibitor therapy and has relapsed. 83. The method according to any one of embodiments 74 to 78, wherein the patient has previously received JAK inhibitor therapy and 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. 85. The method according to embodiment 84, wherein the telomerase inhibitor is imetelstat. 86. The method according to embodiment 85, wherein the imetelstat is imetelstat sodium. 87. The method according to any one of embodiments 74 to 86, further comprising obtaining a sample containing DNA from a patient. 88. 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 the patient, Obtaining bone marrow samples, peripheral blood samples, or combinations thereof, The method according to embodiment 88, comprising isolating DNA from a bone marrow sample, a peripheral blood sample, or a combination thereof. 90. The step of obtaining a sample from the patient, Obtaining bone marrow samples from patients, Isolating cells from bone marrow samples, The method according to embodiment 88, comprising extracting DNA from isolated cells. 91. The step of obtaining a sample from the patient, Obtaining peripheral blood samples from patients, Isolating cells from peripheral blood samples, The method according to embodiment 88, comprising extracting DNA from isolated cells. 92. A method for selecting patients who are most likely to benefit from treatment with telomerase inhibitors, This involves examining a patient for their average relative telomere length by analyzing the relative length of telomere nucleic acids of target cells present in a biological sample derived from the patient. The procedure includes selecting a patient if the patient has an average relative telomere length of target cells present in a patient-derived biological sample that is determined to be within the 50th percentile of a relative telomere length range determined from one or more known criteria, The selected patients are most likely to benefit from treatment with telomerase inhibitors. 93. The method according to embodiment 92, wherein the patient has myelofibrosis. 94. The method according to aspect 93, wherein the myelofibrosis is primary myelofibrosis. 95. The method according to aspect 93, wherein the myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF). 96. The method according to embodiment 93, wherein the myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF). 97. The method according to any of embodiments 92 to 96, wherein the patient has not previously received JAK inhibitor therapy. 98. The patient, I have previously received JAK inhibitor therapy. Previously received JAK inhibitor therapy and failed JAK inhibitor therapy; or The method according to any one of embodiments 92 to 96, wherein the patient has previously received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 99. The method according to any one of embodiments 92 to 96, wherein the patient has previously received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 100. The method according to any one of embodiments 92 to 96, wherein the patient has previously received JAK inhibitor therapy and has experienced a relapse. 101. The method according to any one of embodiments 92 to 96, wherein the patient has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 102. The method according to any one of embodiments 92 to 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 to 104, further comprising obtaining a sample containing DNA from a 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 bone marrow samples, peripheral blood samples, or combinations thereof, The method according to embodiment 106, comprising isolating DNA from a bone marrow sample, a peripheral blood sample, or a combination thereof. 108. The step of obtaining a sample from the patient, Obtaining bone marrow samples from patients, Isolating cells from bone marrow samples, The method according to embodiment 106, comprising extracting DNA from isolated cells. 109. The step of obtaining a sample from the patient, Obtaining peripheral blood samples from patients, Isolating cells from peripheral blood samples, The method according to embodiment 106, comprising extracting DNA from isolated cells. 110. A method for monitoring the effectiveness of treatment in subjects with myelofibrosis (MF), wherein the method is Measuring the hTERT expression level in biological samples obtained from patients after administration of telomerase inhibitors, This includes comparing the hTERT expression level in a biological sample with the baseline hTERT expression level before administration of a telomerase inhibitor. A reduction of 50% or more in hTERT expression levels in biological samples is a method for identifying subjects who are likely to benefit from treatment with telomerase inhibitors. 111. The method according to embodiment 110, wherein the hTERT expression level measured or evaluated is the hTERT RNA expression level. 112. A method for identifying patients with myelofibrosis (MF) for treatment with telomerase inhibitors, wherein the method is Measuring the hTERT expression level in biological samples obtained from patients after administration of telomerase inhibitors, This includes comparing the hTERT expression level in a biological sample with the baseline hTERT expression level before administration of a telomerase inhibitor. A method for identifying patients who are likely to benefit from telomerase inhibitor treatment by reducing hTERT expression levels in biological samples. 113. The method according to embodiment 112, wherein the reduction in hTERT expression level is 50% or more.

[0159] While certain embodiments have been described in some detail as examples and embodiments for the purpose of clarifying understanding, it is readily apparent in light of the teachings of the present invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0160] Therefore, the above is merely illustrative of the principles of the present invention. Various combinations embodying the principles of the present invention and falling within its spirit and scope may be devised, although not expressly described or indicated herein. Furthermore, all embodiments and conditional language enumerated herein are primarily intended to help the reader understand the principles of the present invention and the concepts to which the inventors contribute to further advancing the art, and should be construed as not being limited to such specifically enumerated embodiments and conditions. Moreover, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed to perform the same function, regardless of structure. Therefore, 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 present invention are embodied in the appended claims.

Claims

[Claim 1] The method described in the specification.

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