Methods of identifying patients who are likely to benefit from telomerase inhibitor treatment

Abstract

The present invention provides methods for identifying or selecting a patient who is most likely to benefit from treatment with a telomerase inhibitor, such as imerostat, by performing a test on the patient that each of JAK2, CALR and MPL does not mutate; and / or a macromolecular risk (HMR) determined based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1 / 2. The patient may have myelofibrosis. The invention also provides methods of treating myelofibrosis, including identifying such patients.

Description

[0001] This application is a divisional application of the invention application with a filing date of July 29, 2019, Chinese application number 201980058284.0, and invention name “Method for Identifying Patients Who May Benefit from Telomerase Inhibitor Treatment”. Cross-references to related patent applications

[0002] This patent application claims priority under 35 U.S.C. 119(e) to 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 contents of which are incorporated herein by reference. Sequence Listing

[0003] This patent application contains a sequence listing, which is submitted electronically in ASCII format, and its contents are incorporated herein by reference in their entirety. The ASCII copy is named Sequence_Listing.txt and is 356KB in size. Technical Field

[0004] This patent application relates to methods for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor by identifying patients who do not have mutations in each of JAK2, CALR, and MPL; and / or have a high molecular risk (HMR) determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The invention also relates to methods for treating myelofibrosis in a subject (i.e., patient) in need thereof with a telomerase inhibitor. introduce

[0005] Myelofibrosis (MF) is a classic BCR-ABL1-negative chronic myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation and dysregulated kinase signaling. Cervantes, Blood, 124(17):2635-2642 (2014). It is also characterized by cytopenias, constitutional symptoms, splenomegaly, and possible transformation to 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 for which the JAK1 / JAK2 inhibitor ruxolitinib is currently approved. Ruxolitinib, a Janus kinase (JAK)-1 and JAK-2 inhibitor, is the first drug licensed in the United States for the treatment of moderate to high-grade myelofibrosis (MF). Pardanani et al., J Blood Cancer; 4(12):e268 (2014). Several other JAK inhibitors are being developed, some of which are currently being tested in phase 3 clinical trials. Same as above. Other treatment options for MF include allo-SCT, hydroxyurea, interferon, lenalidomide and thalidomide. Clinical trials are currently underway in MF to evaluate selective JAK inhibitors, histone deacetylase / DNA methyltransferase inhibitors, PI3K inhibitors, Hedgehog / mammalian target of rapamycin (mTOR) inhibitors, antifibrotic agents, immunomodulators, monoclonal antibodies, and immune checkpoint inhibitors. Shreenivas et al., Expert Insights on Emerging Drugs, 23(1):37-49 (2018).

[0006] Other MPNs include essential thrombocythemia (ET) and polycythemia vera (PV). Cervantes (see below). MF may present de novo (primary MF [PMF]) or after a previous diagnosis of ET or PV (post-ET or post-PVMF). Ibid. According to Cervantes, MF is a clonal proliferation of multipotent hematopoietic stem cells in which the abnormal cell population releases multiple cytokines and growth factors in the bone marrow, causing myelofibrosis and stromal changes, and also colonizes extramedullary organs such as the spleen and liver. Ibid. Myelofibrosis has been associated with mutations in the Janus kinase (JAK) 2 gene (e.g., the V617F mutation), mutations in the thrombopoietin receptor gene (MPL), and mutations in the calreticulin gene (CALR). Ibid. It primarily affects the elderly, and according to Cervantes, “currently, there is no curative treatment other than allogeneic hematopoietic stem cell transplantation (allo-SCT) for a minority of patients.” Ibid.

[0007] In fact, according to Langabeer, “most patients with classic myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, harbor distinct disease-driving mutations in the JAK2, CALR, or MPL genes.” Langabeer, JAK-STAT, 5: e1248011 (2016). These mutations are so-called driver gene mutations. Exemplary driver gene mutations include JAK2 V617F and JAK2 exon 12, MPL exon 10, and CALR exon 9 mutations. Same as above.

[0008] According to Spiegel, in myelofibrosis (MF), driver mutations in JAK2, MPL, or CALR affect survival and progression to blast crisis, with triple-negative status (i.e., unmutated JAK2, MPL, and CALR) conferring the greatest risk. Spiegel et al., Blood Research Adv., 1(20):1729-1738 (2017). Indeed, the absence of JAK2 / MPL / CALR mutations (i.e., triple-negative) is associated with the most unfavorable prognosis. Pardanani et al., Blood Cancer J; 4(12):e268 (2014); see also Tefferi et al., Blood, 124(16):2507-13 (2014). In addition, mutations in high molecular risk (HMR) genes (e.g., ASXL1, EZH2, IDH1 / 2, and SRSF2) are also associated with a poor prognosis. Spiegel et al. Independent of traditional risk factors, the presence of an increasing number of prognostically deleterious / “high molecular risk” mutations (i.e., ASXL1, EZH2, SRSF2, and / or IDH-1 / 2 genes) progressively worsens survival outcomes. Guglielmelli et al., Leukemia, 28(9):1804-10 (2014).

[0009] 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 shortened OS. Spiegel observed that these mutations were associated with a shortened time to treatment failure in patients with myelofibrosis treated with ruxolitinib or molotinib (JAK 1 / 2 inhibitors). Ibid. Similarly, “relative to the clinical features 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 counts (mean 11.0 vs 25 g / dL; P 5.033), a trend that is consistent with reports in other MPN cohorts.” Patel et al., Blood, 126(6):790-797 (2015). Patel et al. observed that splenic response in patients treated with ruxolitinib who had ≥3 mutations was inversely correlated with time to treatment cessation. Driver mutations or triple-negative (JAK2, MPL, CALR) status have been observed in patients with myelofibrosis who discontinued JAK inhibitor therapy. See, e.g., Kuykendall et al. Summary of the invention

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

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

[0013] Another embodiment of the present invention is a method for identifying a myelofibrosis patient who is most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) obtaining a DNA sample from a patient; (b) testing the DNA sample from such a patient for: (i) triple negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) high molecular risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (c) selecting the patient who satisfies the following conditions: (i) is in triple negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) has a high molecular risk (HMR), determined 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] The DNA sample can be obtained by first obtaining 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 comprises: obtaining a bone marrow sample from the patient; isolating cells from the bone marrow sample; and extracting DNA from the isolated cells. In another embodiment, the step of obtaining a DNA sample from a patient comprises: obtaining a peripheral blood sample from the patient; isolating cells (e.g., granulocytes) from the peripheral blood sample; and extracting DNA from the isolated cells.

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

[0016] In any of these methods, the patient may have myelofibrosis. The myelofibrosis may be: primary myelofibrosis; post-polycythemia vera myelofibrosis (post-PV MF); or post-essential thrombocythemia myelofibrosis (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 received "failed" JAK inhibitor therapy (i.e., the disease is resistant to the therapy, or the patient is resistant to the therapy, or the disease has relapsed despite initial response to treatment). In other embodiments, the patient has received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance.

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

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

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

[0020] Another embodiment of the present invention is a method of treating a patient with myelofibrosis with a telomerase inhibitor (e.g., imetelstat or imetelstat sodium), comprising: (i) screening said patient to determine whether such patient: has triple negative status, as determined by the absence of mutations in each of JAK2, CALR, and MPL, and / or has high molecular risk (HMR), as determined by the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (ii) administering the telomerase inhibitor to such patient if such patient is in triple negative status and / or is in high molecular risk (HMR), wherein the triple negative status is determined based on the absence of mutations in any of JAK2, CALR and MPL, and the high molecular risk is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1 / 2. The myelofibrosis may be: primary myelofibrosis, post-polycythemia vera myelofibrosis (post-PV MF) or post-essential thrombocythemia myelofibrosis (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 now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance.

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

[0022] In some embodiments of the method of identifying or selecting patients who are most likely to benefit from telomerase inhibitor treatment, the method further comprises determining the average relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present in the biological sample of the patient. In some embodiments of the method of identifying or selecting patients who are most likely to benefit from telomerase inhibitor treatment, the method further comprises selecting the patient whose average relative telomere length in target cells present in the corresponding biological sample is determined to be the 50th percentile or lower of the relative telomere length range (determined according to one or more known standards). In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, the imetelstat is imetelstat sodium.

[0023] The present invention provides a method for treating a patient with myelofibrosis with a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the patient is in triple negative status (determined 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, the imetelstat is imetelstat sodium.

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

[0025] The present invention provides a method of treating a patient with myelofibrosis with a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the patient has one or more of the following characteristics: (a) the average relative telomere length of target cells present in the biological sample of the individual is determined to be at or below the 50th percentile of a range of relative telomere lengths (determined according to one or more known standards); (b) triple-negative status, which is determined based on the absence of mutations in each of JAK2, CALR, and MPL; and (c) high molecular risk (HMR), which is determined based on the presence of a mutation 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, the imetelstat is imetelstat sodium.

[0026] The present invention provides a method for identifying a subject with myelofibrosis (MF) suitable for treatment with a telomerase inhibitor, the method comprising: measuring the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor; and comparing the hTERT expression level in the biological sample with a baseline hTERT expression level before administration of the telomerase inhibitor; wherein a decrease in the hTERT expression level in the biological sample can identify a patient with an increased likelihood of benefiting from treatment with the telomerase inhibitor.

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

[0028] The present invention provides a method for monitoring the effect of treatment in a subject with myelofibrosis (MF), the method comprising: measuring the hTERT expression level in a biological sample obtained from the patient after administration of a telomerase inhibitor; and comparing the hTERT expression level in the biological sample with the baseline hTERT expression level before administration of the telomerase inhibitor; wherein a 50% or more reduction in the hTERT expression level in the biological sample can identify a subject with an increased likelihood of benefiting from treatment with the telomerase inhibitor. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, the imetelstat is imetelstat sodium.

[0029] The present invention provides a method for selecting patients who are most likely to benefit from telomerase inhibitor treatment, comprising: testing the average relative telomere length of a patient by analyzing the relative length of telomere nucleic acids in target cells present in a biological sample of the patient; and selecting the patient whose average relative telomere length in target cells present in the corresponding biological sample is determined to be at or below the 50th percentile of a relative telomere length range (determined according to one or more known standards), wherein the selected patient is most likely to benefit from telomerase inhibitor treatment.

[0030] The present invention provides a method for identifying patients who are most likely to benefit from telomerase inhibitor treatment, comprising: obtaining a biological sample from a patient; determining an average relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present in the biological sample of the patient; and identifying the patient whose average relative telomere length in the target cells present in the biological sample is determined to be at or below the 50th percentile of a relative telomere length range (determined according to one or more known standards), wherein the identified patient is most likely to benefit from telomerase inhibitor treatment.

[0031] The present invention provides a method for treating a patient with myelofibrosis with a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the average relative telomere length in target cells present in a biological sample of such patient is determined to be at or below the 50th percentile of a relative telomere length range (determined according to one or more known standards). In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, the imetelstat is imetelstat sodium.

[0032] The present invention provides a method for monitoring the effect of treatment in a subject with myelofibrosis (MF), the method comprising measuring the hTERT expression level in a biological sample obtained from the patient after administering a telomerase inhibitor; and comparing the hTERT expression level in the biological sample with the baseline hTERT expression level before the telomerase inhibitor is administered; wherein a 50% or more reduction in the hTERT expression level in the biological sample can identify a subject with an increased likelihood of benefiting from treatment with the telomerase inhibitor. In certain embodiments, the measured or assessed hTERT expression level is an hTERT RNA expression level. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, the imetelstat is imetelstat sodium.

[0033] The present invention provides a method for identifying a myelofibrosis (MF) patient suitable for treatment with a telomerase inhibitor, the method comprising: measuring the hTERT expression level in a biological sample obtained from the patient after administration of a telomerase inhibitor; and comparing the hTERT expression level in the biological sample with a baseline hTERT expression level before administration of the telomerase inhibitor; wherein a decrease in the hTERT expression level in the biological sample can identify a patient with an increased likelihood of benefiting from treatment with the telomerase inhibitor.

[0034] The present invention provides a method for monitoring the effect of treatment in a subject with myelofibrosis (MF), the method comprising: measuring the level of telomerase activity in a biological sample obtained from the patient after administration of a telomerase inhibitor; and comparing the level of telomerase activity in the biological sample with the baseline telomerase activity level before administration of the telomerase inhibitor; wherein a reduction of 50% or more in the level of telomerase activity in the biological sample can identify a subject with an increased likelihood of benefiting from treatment with the telomerase inhibitor. In certain embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, the imetelstat is imetelstat sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The most comprehensive understanding of the foregoing summary and the following detailed description of the invention can be obtained by reading in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show embodiments of the invention. However, it should be understood that the invention is not limited to the precise devices, examples and apparatus shown.

[0036] Figure 1 Shown is a waterfall plot of spleen volume reduction (SVR) at week 24 for the 4.7 mg / kg and 9.4 mg / kg treatment groups in Example 1. SVR is expressed as percent change from baseline.

[0037] Figure 2 Shown is a waterfall chart of 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 expressed as a percent change from baseline.

[0038] Figure 3 The Kaplan-Meier plot of overall survival in the 4.7 mg / kg treatment group was presented according to the mutation status of the JAK2 / MPL / CALR gene (TN vs. non-TN (MUT)). Specifically, Figure 3 Shown are plots of survival probability versus time for patients with triple-negative status (TN) and patients with at least one mutation (MUT).

[0039] Figure 4 The Kaplan-Meier plot of overall survival in the 9.4 mg / kg treatment group was presented according to the mutation status of the JAK2 / MPL / CALR gene (TN vs. non-TN (MUT)). Specifically, Figure 4 Shown are plots of the probability of survival versus time for patients with triple negative status (TN) and patients with at least one mutation (MUT) in the 9.4 mg / kg treatment group.

[0040] Figure 5Kaplan-Meier plots showing overall survival over time in the 9.4 mg / kg and 4.7 mg / kg treatment groups according to patient status.

[0041] Figure 6 The Kaplan-Meier plot of overall survival (OS) in the 9.4 mg / kg treatment group according to the mutation status of the JAK2 / MPL / CALR gene (TN vs. non-TN) is shown. Specifically, Figure 6 Shown is a plot of the probability of survival versus time for patients in the 9.4 mg / kg treatment group with triple negative status (TN) and patients with at least one mutation (non-TN).

[0042] Figure 7 The Kaplan-Meier plot of overall survival (OS) in the 4.7 mg / kg treatment group according to the mutation status of the JAK2 / MPL / CALR gene (TN vs. non-TN) is shown. Specifically, Figure 7 Shown is a plot of the probability of survival versus time for patients in the 4.7 mg / kg treatment group with triple negative status (TN) and patients with at least one mutation (non-TN). Implementation

[0043] The present patent application is based on the discovery that patients with myelofibrosis; patients whose corresponding results are triple negative (i.e., no mutations in each of JAK2, CALR and MPL); and / or patients who belong to the high molecular risk (HMR) category (which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1 / 2) can benefit from treatment with telomerase inhibitors (e.g., imetelstat or imetelstat sodium). Patients with mutations in the ASXL1, EZH1, IDH1 / 2 and SRSF2 genes have an increased risk of early death or leukemic transformation. These patients generally do not benefit from treatment with conventional therapies (e.g., JAK inhibitors). Gisslinger et al., Blood, 128:1931 (2016). Therefore, the fact that these patients benefit from treatment with telomerase inhibitors is unexpected and surprising.

[0044] Accordingly, the present patent application provides a method for identifying patients who are most likely to benefit from treatment with a telomerase inhibitor (e.g., imetelstat). The method comprises testing or identifying a patient to determine whether the patient is: in a triple-negative state, which is determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or in a high molecular risk (HMR), which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The present patent application also provides a method for treating myelofibrosis with a telomerase inhibitor (e.g., imetelstat), which involves identifying patients who meet the following conditions: in a triple-negative state, which is determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or having a high molecular risk (HMR), which is determined 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. Then, the telomerase inhibitor (e.g., imetelstat) is administered to the patient. For the purpose of clarifying the invention and not limiting it, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the invention. A. Definition

[0045] As used herein, additional sex comb-like protein 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2) mutations should include any mutations in these genes that affect the survival and disease progression of patients with myelofibrosis. In addition, IDH1 / 2 used in this article should include IDH1 and IHD2. For exemplary mutations, see the following publications, the contents of which are incorporated herein for disclosure of gene mutations associated with myelofibrosis: Langabeer, JAK-STAT, 5:e1248011 (2016); Cervantes, Blood, 124(17):2635-2642 (2014); Patel et al., Blood, 126(6):790-797 (2015); Spiegel et al., Blood Res, 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 shown below: High molecular risk (HMR) can be determined based on the presence of a mutation in at least one of the following genes: an ASXL1 gene having a nucleic acid sequence such as sequence number 5; an EZH2 gene having a nucleic acid sequence such as sequence number 6; an SRSF2 gene having a nucleic acid sequence such as sequence number 7; an IDH1 gene having a nucleic acid sequence such as sequence number 8; an IDH2 gene having a nucleic acid sequence such as sequence number 9; and combinations thereof.

[0046] 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 , 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 mutations. In some embodiments, the mutation is Q575X mutation, Q588X, Y591X mutation, Y591N mutation, Q592X mutation, S604F mutation, L614F mutation, Q623X mutation, A627G mutation, E635R mutation, T638V mutation, A640G mutation, G646W mutation, G658X mutation, R678K mutation, C687R mutation, C687V mutation, D690G mutation, R693X mutation, Y700X mutation, G704R mutation, G704W mutation, E705X mutation, Q708X mutation, G710E mutation, L721C mutation, E727X mutation, V751L mutation, P763R mutation, Q780X mutation, W780V mutation. mutation, 96X mutation, W796G mutation, V807F mutation, T822H mutation, K825X mutation, S846Q mutation, D855A mutation, C856X mutation, L857R mutation, L885X mutation, L890F mutation, S903I mutation, S970N mutation, Y974X mutation, R965X mutation, G967del mutation, V962A mutation, L992Q mutation, S1028R mutation, Q1039L mutation, R1073C mutation, E1102D mutation, H1153R mutation, S1209I mutation, S1231F mutation, A1312V mutation, F1305W mutation, P1377S mutation, R1415Q mutation and / or I1436M mutation.

[0047] In some embodiments, the target mutations in the EZH2 gene include W60, R63, P312, F145, N182, R288, Q328, Q553, R566, T573, R591, R659, D677, V679, R690, A702, V704, E726, D730 and / or Y733 mutations. In some embodiments, the mutation is a W60X mutation, an R63X mutation, a P312S mutation, a F145S mutation, a N182D mutation, a R288Q mutation, a Q328X mutation, a Q553X mutation, a R566H mutation, a T573I mutation, a R591H mutation, a R659K mutation, a D677H mutation, a V679M mutation, a R690H mutation, an A702V mutation, a V704L mutation, an E726V mutation, a D730X mutation, and / or a Y733X mutation.

[0048] In some embodiments, the mutation of interest in the SRSF2 gene comprises a P95 mutation. In some embodiments, the mutation is a P95H mutation, a P95L mutation, or a P95R mutation.

[0049] In some embodiments, the mutation of interest in the IDH1 / 2 gene includes R132 and / or R140 mutations. In some embodiments, the mutation is R132G mutation, R132H mutation or R140Q mutation.

[0050] In certain embodiments, the mutations of interest include the mutations listed below:

[0051] As used herein, "triple negative status", "triple negative" or "TN" refers to the test results of patients in which no mutations are present in each 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 each of the following genes: a JAK2 gene having a nucleic acid sequence such as sequence number 2; a CALR gene having a nucleic acid sequence such as sequence number 3; and an MPL gene having a nucleic acid sequence such as sequence number 4.

[0052] In certain embodiments, triple negative status includes the absence of mutations in the JAK2 gene, e.g., G335, F556, G571, V617, and / or V625 mutations. For example, triple negative status may include the absence of G335D mutations, F556V mutations, G571S mutations, V617F mutations, and / or V625S mutations in the JAK2 gene.

[0053] In certain embodiments, triple negative status includes the absence of a mutation in the MPL gene, e.g., T119, S204, P222, E230, V285, R321, S505, W515, Y591 and / or R592 mutations. For example, triple negative status may include the absence of 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.

[0054] In certain embodiments, triple negative status includes the absence of mutations in the CALR gene, e.g., L367, K368, E381, K385, and / or E396 mutations. 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.

[0055] In certain embodiments, the mutations of interest include the mutations listed below:

[0056] As used herein, a patient has "failed" JAK inhibitor therapy if the disease is resistant to the therapy, or the patient is resistant to the therapy, or the disease has relapsed despite an initial response to treatment.

[0057] As used herein, the term "about" when referring to a measurable value (e.g., an amount, a time interval, etc.) is meant to encompass variations of ±20% to ±0.1%, preferably ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

[0058] The term "pharmaceutically acceptable salt" refers to an acceptable salt suitable for administration to a patient such as a mammal (a salt containing a counterion with acceptable mammalian safety for a given dosing regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, wherein the salt is derived from various organic and inorganic counterions well known in the art and includes, by way of example only, sodium and the like; when the molecule contains a basic functional group, it also includes salts of organic or inorganic acids, such as hydrochloride and the like. Pharmaceutically acceptable salts of interest include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, theophylline, 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.

[0059] The term "salt thereof" refers to a compound formed when the proton of an acid is replaced by a cation (e.g., a metal cation or an organic cation, etc.). Preferably, the salt is a pharmaceutically acceptable salt. For example, the salt of the compound of the present invention includes a salt in which the compound is protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid is used as the anion component of the salt. The salt of interest includes, but is not limited to, aluminum, ammonium, arginine, barium, benzathine penicillin, calcium, theophylline, ethylenediamine, lithium, magnesium, meglumine, procaine, N-methylglucamine, piperazine, potassium, sodium, tromethamine, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine salts. It should be understood that for any oligonucleotide structure described herein (including the backbone of the internucleotide bond), such oligonucleotides may also include any suitable salt form. In some embodiments, for simplicity, the acidic form of the internucleotide bond is depicted. In some cases, the salt of the target compound is a monovalent cation salt. In some cases, the salt of the target compound is a divalent cation salt. In some cases, the salt of the target compound is a trivalent cation salt. "Solvate" refers to a complex formed by a combination of solvent molecules and molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of the two. 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.

[0060] "Stereoisomers" refer to compounds with the same atomic connectivity but different atomic arrangements in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers and diastereomers. For any group containing one or more substituents disclosed by the present invention, it is, of course, to be understood that the group does not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. All stereoisomers are intended to be included within the scope of the present invention.

[0061] Those skilled in the art recognize that other tautomeric arrangements of the groups described herein are also possible. It should be understood that all tautomeric forms of the subject compounds are encompassed by the structure in which one possible tautomeric arrangement of the groups of the compounds is described, even if not explicitly stated.

[0062] It is intended to include solvates of pharmaceutically acceptable salts of tautomers of stereoisomers of the subject compounds. These are intended to be included within the scope of the present invention.

[0063] Before describing certain embodiments in more detail, it should be understood that the present invention is not limited to certain embodiments described, because there will be differences in actual implementation. It should also be understood that the terms used herein are only used to describe certain embodiments, and are not intended to limit the inventive concept, and the scope of the present invention will only be limited by the appended claims.

[0064] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the range are included within the scope of the present invention. Unless the context clearly specifies otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the present invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or two of those included limits is also included within the present invention.

[0065] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0066] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and the purpose of incorporating by reference into this document is to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by reason of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0067] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of special terminology such as "solely," "only," etc. in connection with reciting claim elements or use of a "negative" limitation.

[0068] Each individual embodiment described and listed herein has hierarchical components and features that can be quickly broken down or combined with features of any of the other several embodiments without departing from the scope and spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. B. Identification of patients most likely to benefit from telomerase inhibitor therapy

[0069] In one aspect, the present invention provides a method for identifying or selecting patients who are most likely to benefit from treatment with a telomerase inhibitor. The method relies on identifying patients in a triple-negative state (patients who do not have mutations in each of the JAK2, CALR, and MPL genes) or patients with a high molecular risk (HMR), which is determined based on the presence of at least one mutation in the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. These triple-negative patients or HMR patients are most likely to benefit from treatment with a telomerase inhibitor (e.g., imetelstat or imetelstat sodium).

[0070] The myelofibrosis may be primary myelofibrosis, post-polycythemia vera myelofibrosis (post-PV MF) or post-essential thrombocythemia myelofibrosis (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 received failed JAK inhibitor therapy (i.e., the disease is resistant to the therapy, or the patient is resistant to the therapy, or although initially responsive to treatment, the disease has recurred). In other embodiments, the patient has previously received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. In another alternative embodiment, the patient has previously received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy.

[0071] In one embodiment, the patient has received JAK inhibitor therapy and the myelofibrosis is resistant to JAK inhibitor therapy. In another embodiment, the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. In another embodiment, the patient has received JAK inhibitor therapy and the patient's corresponding disease has relapsed. In an alternative embodiment, the patient has received JAK inhibitor therapy and has stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0072] In one embodiment, the present invention provides a method for selecting patients who are most likely to benefit from telomerase inhibitor treatment by testing one or more of the following: triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes (i.e., the absence of any mutations). In this embodiment, the patient may also be tested for high molecular risk (HMR), which is determined 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 who are most likely to benefit from telomerase inhibitor treatment by testing for the following: triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes (i.e., the absence of any mutations); and / or high molecular risk (HMR), which is determined 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 a patient who is most likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient for: (a) triple negative status, determined based on the absence of any mutations in the JAK2, CALR, and MPL genes; (b) high molecular risk (HMR), determined 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 that the patient is most likely to benefit from treatment with a telomerase inhibitor.

[0073] Another embodiment of the invention is a method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: a. Perform the following tests on the patient: i. Triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. High molecular risk (HMR), which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, b. Select the patient who meets the following conditions: i. triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and / or ii. high molecular risk (HMR), determined 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 a telomerase inhibitor.

[0074] Another embodiment of the present invention is a method of identifying a patient most likely to benefit from treatment with a telomerase inhibitor, comprising: testing a patient for triple negative status, determined based on the absence of a mutation in each of the JAK2, CALR, and MPL genes; and selecting said patient who satisfies the following criteria: is in triple negative status, determined based on the absence of a mutation in each of the JAK2, CALR, and MPL genes, wherein said selected patient is most likely to benefit from treatment with a telomerase inhibitor. In one embodiment, the method further comprises testing the patient for high molecular risk (HMR), determined based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, and comprises selecting said patient with an HMR.

[0075] In certain embodiments of any of these methods, the triple-negative patient does not have mutations in the coding regions (exons) of the JAK2, CALR, and MPL genes.

[0076] Furthermore, in other embodiments of any of these methods, high molecular risk (HMR) is determined based on the presence of a mutation within the coding region (exon) of at least one of the ASXL1, EZH2, SRSF2, and IDH1 / 2 genes.

[0077] In certain embodiments, high molecular risk (HMR) is determined by detecting the presence of a mutation in ASXL1, EZH2, SRSF2, or IDH1 / 2, or a combination thereof. In some embodiments, the method comprises detecting the presence of a mutation in ASXL1. In some embodiments, the method comprises detecting the presence of a mutation in EZH2. In some embodiments, the method comprises detecting the presence of a mutation in SRSF2. In some embodiments, the method comprises detecting the presence of a mutation in IDH1 / 2. In some embodiments, the method comprises detecting the presence of a mutation in ASXL1 and EZH2. In some embodiments, the method comprises detecting the presence of a mutation in ASXL1 and SRSF2. In some embodiments, the method comprises detecting the presence of a mutation in ASXL1 and IDH1 / 2. In some embodiments, the method comprises detecting the presence of a mutation in EZH2, SRSF2. In some embodiments, the method comprises detecting the presence of a mutation in EZH2 and IDH1 / 2. In some embodiments, the method comprises detecting the presence of a mutation in SRSF2 and IDH1 / 2. In some embodiments, the method comprises detecting the presence of a mutation in ASXL1, EZH2, and SRSF2. In some embodiments, the method comprises detecting the presence of a mutation in ASXL1, EZH2, and IDH1 / 2. In some embodiments, the method comprises detecting the presence of mutations in EZH2, SRSF2, and IDH1 / 2. In some embodiments, the method comprises detecting the presence of mutations in ASXL1, EZH2, SRSF2, and IDH1 / 2. In 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 telomerase inhibitor treatment. In this method, patients in the patient population are screened for mutations in each of the corresponding 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 in which each of the corresponding JAK2, MPL, and CALR genes does not have a typical mutation.

[0078] In certain embodiments, the method further comprises the step of obtaining a patient DNA sample. The patient sample can be collected from a DNA sample obtained from bone marrow, peripheral blood or both. Therefore, in certain embodiments, the method of the present invention comprises obtaining a patient blood sample and separating (extracting) DNA from the patient blood sample. The method may also comprise the step of separating cells (e.g., granulocytes) from the patient blood sample. Similarly, the method of the present invention comprises obtaining a bone marrow sample and separating (extracting) DNA from the bone marrow sample. The method may also comprise the step of separating cells from the patient bone marrow sample.

[0079] The patient DNA sample is tested for the presence or absence of mutations in each of the JAK2, CALR, and MPL genes using conventional techniques. Alternatively, the patient DNA sample is tested for the presence of mutations in at least one of the following genes using conventional techniques: ASXL1, EZH2, SRSF2, and IDH1 / 2. In certain embodiments, the patient DNA sample is tested for: (i) the presence or absence of mutations in each of the JAK2, CALR, and MPL genes; and (ii) the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0080] In certain embodiments, the testing of the DNA sample can be a next generation sequencing assay performed using the Illumina MiSeq platform as disclosed in the following publication: Patel et al., Blood, 126(6):790-797 (2015), the contents of which are incorporated herein as they relate to DNA sample testing. C. Pharmacodynamics (PD)

[0081] The invention is based in part on pharmacodynamic effects demonstrating that, in subjects with myelofibrosis, there is an association between response to telomerase inhibition therapy and a decrease in telomerase hTERT expression levels in the subject relative to baseline levels. In some instances, a higher proportion of subjects who have a clinical response (spleen or symptoms) to telomerase inhibition therapy at week 24 have a 50% or greater decrease in hTERT RNA expression levels compared to subjects who have not responded.

[0082] The present invention provides for the stratification and selection of patients who may benefit from telomerase inhibition therapy for myelofibrosis, and provides methods for monitoring response, relapse and prognosis in subjects undergoing treatment.

[0083] Aspects of the invention include methods of selecting a subject with myelofibrosis (MF) for treatment with a telomerase inhibitor, and methods of treating MF. The invention also provides methods of monitoring the efficacy of a treatment in a subject with MF. In some cases, the pharmacodynamic effect on which an embodiment of the subject method is based is a reduction in hTERT RNA expression of 50% or more, such as 60% or more, 70% or more, 80% or more, or 90% or more.

[0084] The telomerase ribonucleoprotein is composed of components or subunits, two of which are telomerase RNA template (hTR) and telomerase reverse transcriptase protein (hTERT). The hTERT expression level can be assessed, determined and / or measured by any suitable method. A variety of methods can be applied to the amplification, detection and measurement of mRNA of telomerase components or related proteins in body fluids. Methods and assays of interest that may be suitable for use with the subject methods include, but are not limited to, real-time quantitative RT-PCR assays, e.g., TaqMan-based fluorescence methods, immunohistochemistry methods for protein expression, and methods described in the following publications: U.S. Pat. No. 6,607,898; Bieche et al., Clinical Cancer Research, Feb. 1, 2000 (6)(2) 452-459; Terrin et al. (“Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and defines subgroups of patients with the same igVH mutation status and different prognoses.” 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).

[0085] The hTERT expression level can be evaluated or measured in any suitable target cell or biological sample. The target cell can be any suitable cell of the patient, including but not limited to cells in the bone marrow or peripheral blood of the patient. In some cases, the target cell is isolated from a bone marrow sample of the patient. In some cases, the target cell is isolated from a peripheral blood sample of the patient. The target cell may be a granulocyte.

[0086] The level of hTERT RNA expression may be assessed or measured in an RNA sample using any suitable method. The RNA sample can be obtained by first obtaining a bone marrow sample, a peripheral blood sample, or both, and then isolating the RNA from the bone marrow sample, the peripheral blood sample, or both. In one embodiment, the step of obtaining an RNA sample from a patient comprises: 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 comprises: obtaining a peripheral blood sample from the patient; isolating cells (e.g., granulocytes) from the peripheral blood sample; and extracting RNA and / or DNA from the isolated cells. D. Treatment

[0087] Aspects of the invention include methods for treating myelofibrosis in a subject (i.e., patient) who meets the following conditions and has corresponding needs: triple negative status, which is determined based on the absence of any mutation in the JAK2, CALR and MPL genes (i.e., no mutation in these genes or no mutation in these genes); and / or high molecular risk (HMR), which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1 / 2. One embodiment of the invention is a method for treating myelofibrosis in a subject (i.e., patient) who meets the following conditions and has corresponding needs: triple negative status, which is determined based on the absence of any mutation in the JAK2, CALR and MPL genes (i.e., no mutation in these genes or no mutation in these genes). In one embodiment, the myelofibrosis is primary myelofibrosis. In another embodiment, the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). In an alternative embodiment, the myelofibrosis is post-essential thrombocythemia myelofibrosis (post-ET MF).

[0088] In certain embodiments of the method of treatment, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy, and has received "failed" JAK inhibitor therapy (that is, the disease is resistant to the therapy, or the patient is resistant to the therapy, or although initially responsive to treatment, the disease has recurred). In alternative embodiments of the method of treatment, the patient has received JAK inhibitor therapy, and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. In certain embodiments, the method of treatment further includes the use of diphenhydramine (25 to 50 mg) and hydrocortisone (100 to 200 mg) or its equivalent for preventive administration.

[0089] A subject is a mammal in need of cancer treatment. Typically, the subject is a human patient. In some embodiments of the invention, the subject may be a non-human mammal, such as a non-human primate, an animal model (e.g., an animal for drug screening, characterization and evaluation, such as mice and rats) and other mammals. The terms patient, subject and individual used herein are used interchangeably.

[0090] "Treatment" as used herein and as known in the art is a method for obtaining a beneficial or desired result (including a clinical result). For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, relief or improvement of one or more symptoms, alleviation of the degree of disease, stabilization of the disease state (i.e., no deterioration), prevention of the spread of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an extension of the survival period compared to the expected survival period in the absence of treatment. E. Telomerase inhibitors

[0091] The methods of the present invention can be used to identify patients who are most likely to benefit from treatment with any suitable telomerase inhibitor. In addition, any suitable telomerase inhibitor can be used in the target treatment method. In some embodiments, the telomerase inhibitor is an oligonucleotide having telomerase inhibitory activity, in particular an oligonucleotide defined in WO 2005 / 023994 and / or WO 2014 / 088785, the contents of which are incorporated herein by reference in their entirety. In some cases, one or more than one telomerase inhibitor (e.g., two or three telomerase inhibitors) may be administered to a mammal to treat a hematological malignancy. Imetelstat

[0092] In certain embodiments, the telomerase inhibitor is imetelstat, including its tautomers and salts thereof, e.g., pharmaceutically acceptable salts. Imetelstat is a novel telomerase inhibitor that has shown clinical activity in hematological malignancies (Baerlocher et al., NEJM 2015, 373:920-928; Tefferi et al., NEJM 2015, 373:908-919) (as shown below): Wherein "nps" represents a phosphorothioate amino ester bond -NH-P(═O)(SH)-O-, which connects the 3' carbon terminal of one nucleoside to the 5' carbon terminal of the adjacent nucleoside.

[0093] In some cases, the telomerase inhibitor is imetelstat sodium, including its tautomers. Imetelstat sodium is the sodium salt of imetelstat, which is a synthetic lipid-conjugated 13-mer oligonucleotide N3'P5' thioamidate. Imetelstat sodium is a telomerase inhibitor, which is a covalently lipidated 13-mer oligonucleotide complementary to the human telomerase RNA (hTR) template region (as shown below). The chemical name of imetelstat sodium is: DNA, d (3'-amino-3'-deoxy-P-thio) (TAGGGTTAGACAA), 5'-[O-[2-hydroxy-3-(hexadecylamino) propyl] thiophosphate], sodium salt (1:13) (sequence number: 1). Imetelstat sodium cannot work through an antisense mechanism, so there are no side effects commonly observed in such therapies.

[0094] Unless otherwise stated or clear from the context, references to imetelstat herein also include its tautomers and salts thereof, e.g., pharmaceutically acceptable salts. As mentioned above, imetelstat sodium is especially the sodium salt of imetelstat. Unless otherwise stated or clear from the context, references to imetelstat sodium herein also include all its tautomers.

[0095] Imetelstat and imetelstat sodium can be produced, formulated or obtained as described elsewhere (see, e.g., Asai et al., Cancer Research, 63:3931-3939 (2003); Herbert et al., Oncogene, 24:5262-5268 (2005); and Gryaznov, Chemistry and Biodiversity, 7:477-493 (2010)). Unless otherwise stated or clear from the context, references to imetelstat sodium herein also include salts thereof. As previously stated, imetelstat sodium is in particular the sodium salt of imetelstat.

[0096] Imetelstat targets the RNA template of telomerase and inhibits telomerase activity and cell proliferation in various cancer cell lines and tumor xenografts in mice. Phase 1 studies involving patients with breast cancer, non-small cell lung cancer and other solid tumors, multiple myeloma or chronic lymphocytic leukemia provide information on the pharmacokinetic and pharmacodynamic characteristics of the drug. Subsequent Phase 2 studies involving patients with essential thrombocythemia showed that reduced platelet activity was accompanied by a significant reduction in the JAK2 V617F and CALR mutant allele loads. Imetelstat sodium is usually administered intravenously; it is expected that other routes of administration, such as intrathecal administration, intratumoral injection, oral administration, etc., can also be used when implementing the target method. Imetelstat sodium can be administered at a dose equivalent to that commonly used in the clinic. In certain embodiments, imetelstat sodium is administered as described elsewhere herein.

[0097] A particular embodiment is based upon any of the other embodiments wherein imetelstat is limited to imetelstat sodium. F. Pharmaceutical Compositions

[0098] For convenient administration, the telomerase inhibitor (e.g., as described herein) can be formulated into various dosage forms for administration purposes. In some cases, the telomerase inhibitor is administered in the form of a pharmaceutical composition. The carrier or diluent of the pharmaceutical composition must be "acceptable" in terms of compatibility with the other ingredients of the composition and harmlessness to the recipient thereof. The pharmaceutical composition can be a single dosage form suitable for, in particular, oral administration, rectal administration, transdermal administration, parenteral injection or inhalation administration. In some cases, it can be administered by intravenous injection. For example, when preparing the composition in an oral dosage form, for oral liquid preparations (e.g., suspensions, syrups, elixirs, emulsions and solutions), any commonly used pharmaceutical solvents, such as water, ethylene glycol, oil, alcohol, etc., can be used; for powders, pills, capsules and tablets, carriers such as starch, sugar, kaolin, diluents, lubricants, binders, disintegrants, etc. can be used. Tablets and capsules are the most advantageous oral dosage unit forms due to their ease of administration, in which case solid pharmaceutical carriers are obviously used. For parenteral compositions, the carrier generally (at least in large part) comprises sterile water, although other ingredients such as those that aid dissolution may generally be included. For example, an injectable solution may be prepared, wherein the carrier comprises a physiological saline solution, a glucose solution, or a mixture of a physiological saline and a glucose solution. For example, an injectable solution may be prepared, wherein the carrier comprises a physiological saline solution, a glucose solution, or a mixture of a physiological saline and a glucose solution. Injectable solutions containing telomerase inhibitors as described herein may be formulated in oil to prolong effectiveness. Suitable oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerides of long-chain fatty acids, and mixtures of these and other oils. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, etc. may be used. Also included are solid form preparations intended to be converted into liquid form preparations shortly before use. In the composition suitable for transdermal administration, the carrier optionally comprises a penetration enhancer and / or a suitable wetting agent, optionally in combination with a small amount of suitable additives of any nature, wherein the additives do not produce significant harmful effects on the skin. The additives may facilitate administration to the skin and / or may contribute to the preparation of the desired composition. The composition may be administered in various ways, for example, as a transdermal patch, a spot-on, an ointment.

[0099] For the convenience of administration and dosage uniformity, it is particularly advantageous to formulate the above-mentioned pharmaceutical composition into a unit dosage form. As used herein, a unit dosage form refers to a physically discrete unit suitable for a single dose, each unit containing a predetermined amount of active ingredient calculated in an amount that produces the desired therapeutic effect in combination with the necessary pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions, and the like, and separated multiple combinations thereof.

[0100] In order to enhance the solubility and / or stability of the drugs described herein in pharmaceutical compositions, it may be advantageous to use α-, β- or γ-cyclodextrin or derivatives thereof, particularly hydroxyalkyl-substituted cyclodextrins, e.g., 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Similarly, cosolvents (e.g., alcohols) may also improve the solubility and / or stability of the telomerase inhibitor in pharmaceutical compositions.

[0101] Depending on the mode of administration, the pharmaceutical composition preferably comprises 0.05 to 99% (weight ratio), more preferably 0.1 to 70% (weight ratio), even more preferably 0.1 to 50% (weight ratio) of the telomerase inhibitor described herein, and 1 to 99.95% (weight ratio), more preferably 30 to 99.9% (weight ratio), even more preferably 50 to 99.9% (weight ratio) of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition. G. Dosage and Dosing Regimen

[0102] The dosing frequency can be any frequency that reduces the severity of myelofibrosis symptoms and does not produce significant toxicity to the subject. For example, the dosing frequency can be about once every two months to about once a week, or about once a month to about twice a month, or about once every six weeks, about once every 5 weeks, or about once every 4 weeks, or about once every 3 weeks, or about once every 2 weeks, or about once a week. During the treatment period, the dosing frequency can remain constant or can be variable. The treatment process using a composition containing one or more telomerase inhibitors can include a rest period. For example, a composition containing a telomerase inhibitor can be administered once a week over a three-week period, then enter a two-week rest period, and such a regimen can be repeated multiple times. Just as the effective amount, various factors may affect the actual dosing frequency for a specific application. For example, the dosing frequency may need to be increased or decreased depending on the effective amount, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of myelofibrosis and related symptoms.

[0103] The effective duration of administering a composition containing a telomerase inhibitor (e.g., imetelstat or imetelstat sodium) can be any duration that reduces the severity of myelofibrosis symptoms (e.g., as described herein) and does not produce significant toxicity to the subject. Therefore, the effective duration can be 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 18 months). In general, the effective duration for treating myelofibrosis can be two months to twenty months. In some cases, the effective duration can be the time a single subject survives. Various factors may affect the actual effective duration for a specific treatment. For example, the effective duration may vary with the frequency of administration, effective amount, the use of a variety of therapeutic agents, the route of administration, and the severity of myelofibrosis and related symptoms.

[0104] In some 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 reduced. For example, a biopsy technique can be used to assess the severity of myelofibrosis symptoms (e.g., as described herein).

[0105] The telomerase inhibitor used in the subject method can be administered at any therapeutically effective dose, such as a dose comparable to that commonly used in the clinic. Specific dosing regimens (e.g., recommended effective doses) for known and approved anticancer drugs are known to physicians and are given, for example, in product instructions, which can be obtained by referring to the following publications: Physician's Desk Reference, 2003, 57th Edition, Medical Economics Company, Inc., Oradell, NJ; Goodman Gilman's Pharmacological Basis of Therapeutics, 2001, 10th Edition, McGraw-Hill, New York; and / or can be obtained from the Federal Drug Administration and / or discussed in the medical literature.

[0106] In some aspects, the dose of telomerase inhibitor imetelstat sodium administered to the subject is about 1.0 mg / kg to about 13.0 mg / kg. In other aspects, the dose of telomerase inhibitor is about 4.5 mg / kg to about 11.7 mg / kg or about 6.0 mg / kg to about 11.7 mg / kg or about 6.5 mg / kg to about 11.7 mg / kg. In some embodiments, the dose of telomerase inhibitor includes at least about 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2mg / 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, 9.2mg / 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, 11.2 Any one of 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg or 13 mg / kg.

[0107] In some embodiments, the effective amount of a telomerase inhibitor administered to the individual comprises at least about any of 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In some embodiments, the effective amount of a telomerase inhibitor administered to the individual is about any of 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 6.5 mg / kg, 7.5 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In various embodiments, the effective amount of a telomerase inhibitor administered to the individual comprises less than about any of 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 of a telomerase inhibitor.

[0108] Exemplary dosing frequencies suitable for pharmaceutical compositions including telomerase inhibitors include, but are not limited to, once daily; once every other day; twice a week; three times a week; once a week without interruption; once a week, three weeks of administration; once every three weeks; once every two weeks; once a week, two weeks of administration. In some embodiments, the pharmaceutical composition is administered approximately once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. In some embodiments, the composition is administered at least about 1x, 2x, 3x, 4x, 5x, 6x, or 7x (i.e., every day) per week, or any of three times a day, twice a day. In some embodiments, the time interval between each administration is less than any of about 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 time interval between each administration is greater than about any one of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there is no interruption in the administration schedule. In some embodiments, the time interval between each administration is no more than about one week.

[0109] Telomerase inhibitors such as imetelstat (e.g., imetelstat sodium) can be administered by any appropriate method. For example, telomerase inhibitors such as imetelstat (e.g., imetelstat sodium) can be administered intravenously once every 4 weeks for a period of time (e.g., 1, 2, 3, 4, 5 hours). In some embodiments, imetelstat is administered intravenously once a week at a dosage of 7-10 mg / kg for about 2 hours at a time. In certain embodiments, imetelstat is administered intravenously once every 3 weeks at a dosage of about 0.5-9.4 mg / kg for about 2 hours at a time. In one embodiment, imetelstat is administered intravenously once every 4 weeks at a dosage of 0.5-5 mg / kg for about 2 hours at a time. In one embodiment, imetelstat is administered intravenously once every 3 weeks at a dosage of about 2.5-10 mg / kg for about 2 hours at a time. Alternatively, imetelstat is administered intravenously once every 4 weeks at a dosage of about 0.5-9.4 mg / kg for about 2 hours at a time.

[0110] In certain embodiments of the method, imetelstat is administered for 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 dosing cycles, each cycle comprising: intravenous administration of about 7-10 mg / kg imetelstat once every three weeks; intravenous administration of about 7-10 mg / kg imetelstat once a week for three weeks; intravenous administration of about 2.5-10 mg / kg imetelstat once every three weeks; or intravenous administration of about 0.5-9.4 mg / kg imetelstat once every three weeks. In some cases, each dosing cycle comprises intravenous administration of about 7-10 mg / kg imetelstat once every three weeks. In some cases, each dosing cycle comprises intravenous administration of about 9.4 mg / kg imetelstat approximately once every three weeks.

[0111] In one embodiment of the present invention, imetelstat is administered intravenously at a dose of about 7-10 mg / kg imetelstat once every three weeks after prophylactic administration with antihistamines, corticosteroids, or both. In other embodiments, imetelstat is administered intravenously at a dose of about 9.4 mg / kg, or about 7.0 mg / kg to 9.8 mg / kg imetelstat once every three weeks after prophylactic administration with antihistamines, corticosteroids, or both.

[0112] In certain embodiments, imetelstat is administered at a dose of about 7.5 mg / kg, or about 7.0 mg / kg to 7.7 mg / kg, once every three weeks for at least three cycles, and then the dose is increased. In certain embodiments, the dose of imetelstat may be increased to about 9.4 mg / kg, or about 8.8 mg / kg to about 9.6 mg / kg, provided that the ANC and platelet nadir have not decreased to about 1.5 x 10 9 / L to about 75x 109 / L, and no grade ≥3 non-hematological toxicity occurred.

[0113] It will be appreciated that cancer treatment sometimes involves multiple "rounds" or "cycles" of administration of a drug, wherein each cycle comprises one or more administrations of the drug according to a specified schedule (e.g., administration on three consecutive days every three weeks; administration once a week; etc.). For example, an anticancer drug may be administered for 1 to 8 cycles or longer. When more than one drug (e.g., two drugs) is administered to a subject, each drug may be administered according to its own schedule (e.g., once a week; once every three weeks; etc.). Obviously, the administration of drugs (even drugs administered in different cycles) may be coordinated so that two drugs are administered on the same day at least sometimes, or so that the drugs are administered for several consecutive days at least sometimes.

[0114] In certain embodiments, the imetelstat may be administered in accordance with a regimen involving a dose reduction. In one embodiment, the patient is initially administered once every three weeks at a dose of about 9.4 mg / kg, then the dose is changed to once every three weeks at a dose of about 7.5 mg / kg, and then the dose is changed to once every three weeks at a dose of about 6.0 mg / kg.

[0115] As is known in the art, if toxicity is observed, or for the convenience of the patient, treatment with a cancer therapeutic agent may be temporarily suspended and then resumed without departing from the scope of the present invention.

[0116] Aspects of the target method include identifying or selecting the patient who is most likely to benefit from treatment based on the relative telomere length in the patient's target cells (e.g., as described herein). The target cell can be any suitable cell of the patient, including but not limited to cells in the bone marrow or peripheral blood of the patient. In some cases, the target cell is isolated from a bone marrow sample of the patient. In some cases, the target cell is isolated from a peripheral blood sample of the patient. The target cell may be a granulocyte. In some cases, each of the patient's Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes is not mutated; and the patient's target cells have particularly short telomere lengths. The short telomere length used herein refers to a telomere length that is less than or equal to the median or average telomere length compared to a suitable control (e.g., one or more known standards described herein). Therefore, the subject method may further include determining relative telomere length by analyzing the relative length of telomere nucleic acid in target cells present in the biological sample of the individual; selecting an individual who will benefit from treatment with a telomerase inhibitor when the average relative telomere length in the target cells present in the biological sample of the individual is determined to be the 50th percentile or lower of a range of relative telomere lengths (determined according to one or more known standards), for example, determined to be the 45th percentile or lower, the 40th percentile or lower, the 35th percentile or lower, the 30th percentile or lower, the 25th percentile or lower, the 20th percentile or lower, or even lower of a range of relative telomere lengths (determined according to one or more known standards).

[0117] In some cases of the methods, the one or more known standards are telomere length ranges set based on a plurality of naturally occurring target cells (e.g., as described herein) from a plurality of individuals diagnosed with the disease. In some cases of the methods, the one or more known standards are characterized cell lines. "Characterized cell line" refers to a cell line in which the relative telomere nucleic acids of the cells are known and relatively constant.

[0118] In some embodiments, the telomere length in the cancer cells present in the biological sample is determined to be less than or equal to the median or mean telomere length. In some embodiments, the telomere length in the cancer cells present in the biological sample is determined to be the 50th percentile or lower, the 40th percentile or lower, the 35th percentile or lower, the 30th percentile or lower, the 25th percentile or lower, the 20th percentile or lower, the 15th percentile or lower, the 10th percentile or lower, or the 5th percentile or lower of the relative telomere length range (determined according to one or more known standards).

[0119] Telomere length in target cells can be determined by any suitable assay, including but not limited to qPCR, telo-FISH or Southern Blot assays, as described by Bassett et al. in U.S. Pat. No. 9,200,327. In one aspect, telomere length can be determined by measuring the average length of terminal restriction fragments (TRFs). The TRF is defined as the length (usually the average length) of the fragments obtained by completely digesting genomic DNA with a restriction enzyme that does not cut the nucleic acid in the telomere sequence. In some cases, the DNA is digested by a restriction enzyme that frequently cuts in genomic DNA but does not cut in the telomere sequence. In some cases, the restriction enzyme has a four-base recognition sequence (e.g., AluI, HinfI, RsaI and Sau3A1) and can be used alone or in combination. The resulting terminal restriction fragment contains telomeric repeats and subtelomeric DNA. Subtelomeric DNA is a DNA sequence adjacent to the tandem repeats of the telomere sequence and contains telomeric repeats interspersed with variable telomere-like sequences. The digested DNA is separated by electrophoresis and imprinted on a support (e.g., film). The fragment containing the telomeric sequence is detected by hybridizing a probe (i.e., labeled repetitive sequence) with the film. After visualization of the fragment containing telomeres, the average length of the terminal restriction fragments can be calculated (Harley, CB et al., Nature, 345 (6274): 458-60 (1990), which is incorporated herein by reference). The TRF estimation performed by Southern blotting gives the distribution of telomere length in the cell or tissue, and therefore also gives the median and mean values ​​of telomere length in all cells.

[0120] On the other hand, telomere length can be measured by flow cytometry (Hultdin, M. et al., Nucleic Acids Research, 26: 3651-3656 (1998); Rufer, N. et al., Nature: Biotechnology, 16: 743-747 (1998), which are incorporated herein by reference). Flow cytometry is a variant of FISH technology. If the starting material is a tissue, a cell suspension is usually prepared by mechanical separation and / or protease treatment. The cells are fixed with a fixative and hybridized with a telomere sequence-specific probe (preferably a PNA probe) labeled with a fluorescent marker. After hybridization, the cells are washed and then analyzed by FACS. After appropriately subtracting background fluorescence, the fluorescence signal of the cells in Go / G1 is measured. This technique is suitable for rapid estimation of telomere length of a large number of samples. Similar to TRF, telomere length is the average length of the telomeres in the cells.

[0121] In other aspects, the median or average length of the telomeres in cells within a biological sample is determined by quantitative PCR (qPCR) or telomere fluorescence in situ hybridization (telo-FISH). In qPCR, a DNA binding dye binds to all double-stranded DNA that causes the dye to fluoresce. The increase in DNA products during the PCR reaction increases the fluorescence intensity and is measured in each cycle of the PCR reaction. This can quantify the DNA concentration. The relative concentration of the DNA present in the exponential phase of the reaction is determined by plotting the relationship between the fluorescence level and the number of PCR cycles on a semi-logarithmic scale. A threshold for detecting fluorescence above background is determined. The number of cycles experienced when the fluorescence from the sample reaches the threshold is called the cycle threshold (Ct). Since the number of DNA theoretically doubles in each cycle of the exponential phase, the relative number of DNA can be calculated. The baseline is the corresponding value of the initial cycle of PCR, where the fluorescence signal is almost unchanged.

[0122] In some aspects, telomere length is determined by telo-FISH. In this method, cells are fixed and hybridized with probes conjugated to fluorescent markers (e.g., Cy-3, fluorescein, rhodamine, etc.). The probe used for this method is an oligonucleotide designed for specific hybridization with telomere sequences. Typically, the probe has a length of 8 or more nucleotides, such as 12-20 or more nucleotides. On the one hand, the probe is an oligonucleotide comprising naturally occurring nucleotides. On the one hand, the probe is a peptide nucleic acid, which has a higher Tm than similar natural sequences, thus allowing the use of more stringent hybridization conditions. Cells can be treated with reagents such as colcemid to induce metaphase arrest in the cell cycle, thereby providing metaphase chromosomes for hybridization and analysis. In some embodiments, cell DNA can also be stained with the fluorescent dye 4', 6-diamidino-2-phenylindole (DAPI).

[0123] The digital image of the complete metaphase chromosome is obtained, and the fluorescence intensity of the probe hybridized with the telomere is quantified. In addition to measuring the average telomere length or the median telomere length of the cell, this method can also measure the telomere length of individual chromosomes and avoid problems related to the presence of subtelomeric DNA (Zjilmans, JM et al., Proceedings of the National Academy of Sciences of the United States of America 94:7423-7428 (1997); Blasco, MA et al., Cell 91:25-34 (1997); incorporated by reference). The intensity of the fluorescent signal is related to the length of the telomere, and the brighter the fluorescent signal, the longer the telomere.

[0124] In certain embodiments, the present invention relates to a telomerase inhibitor for use in a method of treating myelofibrosis, the method comprising: Identification of patients most likely to benefit from telomerase inhibitor therapy involves testing patients for: (a) triple-negative status, which is determined based on the absence of any mutations in the JAK2, CALR, and MPL genes; (b) high molecular risk (HMR), which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; or (c) both; wherein the presence of (a), (b) or (c) indicates that the patient is most likely to benefit from treatment with a telomerase inhibitor, and an effective amount of a telomerase inhibitor is administered to the patient. In certain embodiments, the invention relates to a telomerase inhibitor for use in the method defined in any of the other embodiments.

[0125] Another embodiment of the present invention is a telomerase inhibitor for treating myelofibrosis, comprising: (a) screening patients to determine whether such patients are: triple negative, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or have a high molecular risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) administering the telomerase inhibitor to patients who meet the following conditions: triple negative, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or have a high molecular risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In one embodiment, the use comprises screening the patient for triple negative status, wherein the triple negative status is determined based on the absence of mutations in each of JAK2, CALR, and MPL. Another embodiment of the present invention is a telomerase inhibitor for treating myelofibrosis, comprising: (a) screening a patient to determine whether such patient: is in a triple negative state, which is determined based on the absence of mutations in each of JAK2, CALR and MPL; and (b) if such patient is in a triple negative state, administering the telomerase inhibitor to the patient.

[0126] Another embodiment of the present invention is the use of a telomerase inhibitor for treating myelofibrosis, comprising: (a) screening a patient to determine whether such patient is: in triple negative status, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or has a high molecular risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) administering the telomerase inhibitor to a patient who meets the following conditions: in triple negative status, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or has a high molecular risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In one embodiment, the use comprises screening the patient for triple negative status, the triple negative status being determined based on the absence of mutations in each of JAK2, CALR, and MPL. Another embodiment of the present invention is the use of a telomerase inhibitor for treating myelofibrosis, comprising: (a) screening a patient to determine whether such patient: is in a triple negative state, which is determined based on the absence of mutations in each of JAK2, CALR and MPL; and (b) if such patient is in a triple negative state, administering the telomerase inhibitor to the patient.

[0127] In certain embodiments, triple negative status can be determined based on the absence of mutations in each of the following genes: the JAK2 gene having a nucleic acid sequence of sequence number 2; the CALR gene having a nucleic acid sequence of sequence number 3; and the MPL gene 4 having a nucleic acid sequence of sequence number 4. In other embodiments, triple negative status can be determined based on the absence of mutations in each of the following: sequence number: 2; CALR and MPL. In alternative embodiments, triple negative status can be determined based on the absence of mutations in each of the following: JAK2, sequence number: 3, and MPL. In alternative embodiments, triple negative status can be determined based on the absence of mutations in each of the following: JAK2, CALR, and sequence number: 4.

[0128] In other embodiments of the present invention, high molecular risk (HMR) can be determined based on the presence of a mutation in at least one of the following genes: the ASXL1 gene having a nucleic acid sequence of sequence number 5; the EZH2 gene having a nucleic acid sequence of sequence number 6; the SRSF2 gene having a nucleic acid sequence of sequence number 7; the IDH1 gene having a nucleic acid sequence of sequence number 8; the IDH2 gene having a nucleic acid sequence of sequence number 9; and combinations thereof.

[0129] In other embodiments of the present invention, telomerase activity and hTERT expression levels in biological samples obtained from patients can be determined to evaluate pharmacodynamic effects and / or monitor patients receiving telomerase inhibition therapy. The telomerase activity can be measured using TRAP (Telomere Repeat Amplification Program) telomerase activity assay. The hTERT expression level can be determined by measuring the hTERT RNA expression level in the cells in the biological sample using Northern blotting or sequence analysis of gene expression (SAGE) or other methods.

[0130] In certain embodiments, the invention relates to a telomerase inhibitor for use in the treatment of myelofibrosis as defined in any other embodiment.

[0131] In certain embodiments, the invention relates to the use of a telomerase inhibitor for the treatment of myelofibrosis as defined in any other embodiment. Other embodiments

[0132] Other purpose embodiments are listed in the following clauses:

[0133] Item 1. A method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing a patient for triple negative status, which is determined based on the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes; as well as (b) selecting the patient in triple negative status, wherein the triple negative status is based on JAK2, CALR and MPL genes, The selected patients are most likely to benefit from treatment with a telomerase inhibitor.

[0134] Clause 2. A method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: (c) The patient is tested for the following: i. Triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. High molecular risk (HMR), which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, (d) selecting said patient who is: i. Triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or ii. High molecular risk (HMR), which is determined 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 a telomerase inhibitor.

[0135] Clause 3. A method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: (e) obtaining DNA samples from patients; (f) performing a triple negative status test on the DNA sample derived from the patient, based on JAK2, The absence of mutations in each of CALR and MPL is determined; and (g) selecting the patient who meets the following conditions, being in triple negative status, which is determined 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 a telomerase inhibitor.

[0136] Clause 4. A method of identifying patients most likely to benefit from treatment with a telomerase inhibitor, comprising: (h) obtaining DNA samples from patients; (i) subjecting the DNA sample derived from the patient to the following tests: i. Triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and / or ii. High molecular risk (HMR), which is determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (j) selecting the patient who meets the following conditions: i. Triple negative status, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and / or ii. High molecular risk (HMR), which is determined 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 a telomerase inhibitor.

[0137] Clause 5. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the patient is determined to be in triple negative status, wherein the triple negative status comprises the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes.

[0138] Clause 6. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the patient is determined to be at high molecular risk (HMR), wherein a mutation is present in a gene with an HMR comprising at least one selected from the group consisting of additional sex comb-like protein 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0139] Clause 7. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the mean relative telomere length of cells present in a biological sample of the patient is determined to be at or below the 50th percentile of a range of relative telomere lengths (determined according to one or more known standards).

[0140] Clause 8. Use of a telomerase inhibitor in the manufacture of a medicament for treating a patient with myelofibrosis, wherein the patient is determined to be in triple negative status, wherein the triple negative status comprises the absence of a mutation in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes.

[0141] Clause 9. Use of a telomerase inhibitor in the manufacture of a medicament for treating a patient with myelofibrosis, wherein the patient is determined to have a high molecular risk (HMR), wherein the HMR comprises the presence of a mutation in at least one gene selected from the group consisting of: additional sex comb-like protein 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0142] Clause 10. Use of a telomerase inhibitor in the manufacture of a medicament for treating a patient with myelofibrosis, wherein the mean relative telomere length of cells present in a biological sample of the patient is determined to be at or below the 50th percentile of a range of relative telomere lengths determined according to one or more known standards.

[0143] The following examples are provided for illustration only and are not intended to be limiting. Example Example 1 : Imetelstat is for patients whose disease has relapsed or is resistant to Janus kinase (JAK) inhibitor therapy. Effective treatment for patients with intermediate-2 (Int-2) or high-risk myelofibrosis (MF) introduce

[0144] Imetelstat, a 13-mer oligonucleotide that specifically targets the RNA template of human telomerase, is a potent competitive inhibitor of telomerase enzymatic activity (Asai et al., Cancer Research, 2003; Herbert, Oncogene, 2005). Clinical activity and an acceptable safety profile were reported in a pilot study of 33 patients with intermediate-2 (int-2) or high-risk myelofibrosis (MF), 48% of whom had previously received Janus kinase inhibitor (JAKi) therapy (Tefferi, New England Journal of Medicine, 2015). This example provides results from a phase 2 clinical study of imetelstat sodium in patients with myelofibrosis (MF) at two dose levels. method

[0145] A randomized, multicenter, Phase 2 study of two doses of imetelstat sodium (9.4 mg / kg or 4.7 mg / kg, intravenously, every 3 weeks) was conducted in adults who met the following criteria: Dynamic International Prognostic Scoring System (DIPSS) score of int-2; or high-risk MF that had relapsed / resistant to prior JAKi therapy (i.e., splenomegaly that did not decrease within 12 weeks of initiation of JAK inhibitor (“JAKi”) therapy; or splenomegaly that worsened at any time after initiation of said therapy). A diagnosis of idiopathic, post-essential thrombocythaemia myelofibrosis, or post-polycythaemia vera myelofibrosis was required; other eligibility criteria included measurable splenomegaly (determined by magnetic resonance imaging [MRI]), systemic symptoms associated with active MF, and platelet counts ≥75 x 10 9 / L. The primary endpoints were: spleen response rate (percent spleen volume reduction [SVR] measured by MRI at week 24 ≥35%); and symptom response rate (reduction in total symptom score [TSS] obtained by the Myelofibrosis Symptom Assessment Form (MFSAF) v2 at week 24 ≥50%). Key secondary endpoints included safety, overall survival (OS), treatment response, molecular response, and pharmacokinetic and pharmacodynamic relationships. result

[0146] A total of 107 patients were enrolled at 55 institutions (48 patients received 4.7 mg / kg; 59 patients received 9.4 mg / kg). Baseline characteristics are shown in Table 1 below. In addition, the median JAKi treatment duration was 23 (0.9-89.7) months, and the median platelet count was 147 x 10 9Patients with triple-negative results (TN; i.e., no mutations in JAK2, MPL, or CALR) accounted for 24.8% of the total number of patients, while 67.6% of patients were considered to have high molecular risk (HMR; i.e., ≥1 mutation in ASXL1, EZH2, SRSF2, or IDH1 / 2).

[0147] At the end of the initial clinical study, the median study duration was 22.6 months (range, 0.2-27.4 months); the median treatment duration was 6.2 months (range, 0.0-27.2 months). IRC confirmed that 6 patients (10.2%) in the 9.4 mg / kg treatment group had splenic reactions based on MRI.

[0148] At the end of the clinical study, patients were followed up for a median of 22.6 (0.2-27.4) months, including a median treatment duration of 6.2 (0.0-27.2) months. The median duration of treatment was longer in the 9.4 mg / kg group (7.7 months) than in the 4.7 mg / kg group. Based on MRI, 6 patients (10.2%) in the 9.4 mg / kg group had splenic reactions, while no patients in the 4.7 mg / kg group had reactions (see Table 1). Figure 1 19 patients (32%) in the 9.4 mg / kg group and 3 patients (6%) in the 4.7 mg / kg group experienced symptomatic response (TSS reduction ≥ 50%) (see Figure 2 ).

[0149] At the end of the first clinical study, the median OS was not reached in the 9.4 mg / kg treatment group, while the median OS was 19.9 months in the 4.7 mg / kg treatment group. The 18-month survival rates were 76.7% and 62.9% in the 9.4 mg / kg and 4.7 mg / kg treatment groups, respectively. During dose escalation, a sensitivity analysis was performed on patients who were examined for subsequent JAKi treatment or stem cell transplantation that produced similar results. In the 9.4 mg / kg treatment group, an association was observed between TN patients and OS (TN patients had not yet reached the median OS, and non-TN patients had a median OS of 23.6 months). Patients carrying 1 HMR mutation (ASXL1, EZH2, SRSF2, or IDH1 / 2) had a higher spleen response rate.

[0150] In the 9.4 mg / kg treatment group, the most common adverse events (all grades) during treatment were thrombocytopenia (49%), anemia (44%), neutropenia (36%), and nausea (34%); in the 4.7 mg / kg treatment group, the most common adverse events during treatment were diarrhea (38%), nausea (31%), anemia (31%), and thrombocytopenia (23%). Grade 3 / 4 neutropenia and thrombocytopenia occurred more frequently in the 9.4 mg / kg treatment group (34% and 42%, respectively) compared to the 4.7 mg / kg treatment group (13% and 29%, respectively); most cytopenias resolved within 4 weeks. Grade 3 / 4 LFT elevations were observed in 7 patients during the study. No imetelstat-related liver toxicity confirmed by the independent liver review committee was observed.

[0151] At the end of the second clinical study, patients were followed up with a median follow-up time of 27.4 (0.2-33.0) months, including a median treatment time of 26.9 (0.1-118.1) weeks. The median treatment duration of the 9.4 mg / kg treatment group (33.3 weeks) was longer than that of the 4.7 mg / kg treatment group (23.9 weeks). The relevant study of the 4.7 mg / kg treatment group was terminated early, affecting the duration of treatment. In the 9.4 mg / kg treatment group, the median OS (95% confidence interval) was 29.9 months (22.8, NE) (NE means not estimable), and this median was reached at the end of the second clinical study. Triple Negative and OS

[0152] Subjects were grouped according to the mutation status of the JAK2 / MPL / CALR genes - triple negative (TN, no mutation in each of the JAK2 / MPL / CALR genes) and non-TN (mutation in any of the JAK2 / MPL / CALR genes). In the 9.4 mg / kg treatment group, the median OS of TN subjects (95% confidence interval) (23.2, NE) was not estimable (NE), and the median OS of non-TN subjects (95% confidence interval) (20.7, NE) was 23.6 months, while in the 4.7 mg / kg treatment group, the median OS (95% confidence interval) of TN subjects and non-TN subjects were 22.3 (17, NE) and 20.3 (18.3, NE), respectively. In the 9.4 mg / kg treatment group, the mortality rate of the triple negative (TN) group was lower than that of the non-TN group (see Table 2, Figure 3 and 4 ).

[0153] At the end of the second clinical study, in the 9.4 mg / kg treatment group, the mortality rate in the triple negative (TN) group was lower than that in the non-TN group (see Table 3, Figure 6 and 7 ). Table 3: Overall survival according to JAK2 / MPL / CALR gene mutation status *HR = hazard ratio Relationship between negative and positive responses on Wednesday 24th

[0154] In the 9.4 mg / kg treatment 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: Baseline JAK2 / CALR / MPL mutation status according to clinical response Relationship between molecular risk and response at week 24

[0155] In the 9.4 mg / kg treatment group, higher response rates (SVR or TSS) were observed in the low molecular risk (LMR) group or high molecular risk (HMR) group with only one mutation (mut) compared with the HMR group with more than one mutation (see Table 5). Table 5: Relationship between molecular risk and response at week 24

[0156] In subjects who received the 9.4 mg / kg dose, the following factors were observed to be associated with clinical response or OS: Triple Negative (TN): Enhanced response (SVR or TSS) in TN subjects. Median OS in the TN group was not estimable, while median OS in the non-TN group = 23.6 months; and Molecular Risk: Responses (SVR or TSS) were enhanced in HMR subjects with only 1 mutation, and responses were observed in HMR patients with more than 1 mutation who received 9.4 mg / kg of imetelstat. Example 2 Baseline telomere length (TL) and overall survival (OS)

[0157] Subjects were grouped according to the median of baseline TL. In the 9.4 mg / kg treatment group, at the end of the first clinical study, the median OS for subjects with a shorter baseline TL (<= median) was not estimable (95% confidence interval) (23.2, NE); while for subjects with a longer baseline TL (> median), the median OS was 22.8 months (16.2, NE) (Table 6). In the 4.7 mg / kg treatment group, the median OS (95% confidence interval) for subjects with a shorter baseline TL and subjects with a longer TL was 20.3 (17.2, NE) months and 22.3 (16.6, NE) months, respectively (Table 6).

[0158] In the 9.4 mg / kg treatment group, a trend toward better OS was observed in subjects with shorter baseline TL (i.e., baseline TL less than or equal to the median TL). Table 6: Corresponding overall survival according to baseline telomere length (TL) grouped according to median value Relationship between baseline TL and response at week 24

[0159] Baseline Telomere Length (TL): At Week 24, SVR or TSS responses were enhanced in subjects with shorter baseline TL (<= median). Spleen responses occurred in 17.3% (5 / 29) of subjects with shorter baseline TL and 4.2% (1 / 24) of subjects with longer baseline TL. TSS responses occurred in 34.5% (10 / 29) of subjects with shorter baseline TL and 25% (6 / 24) of subjects with longer baseline TL.

[0160] At week 24, in the 9.4 mg / kg treatment group, subjects with shorter baseline TL showed higher response rates (SVR or TSS) compared with subjects with longer TL (Table 7). Table 7: Summary of Baseline Telomere Length (TL) by Clinical Response Example 3 Dose-dependent pharmacodynamic (PD) effects Telomerase activity and hTERT were analyzed to evaluate the pharmacodynamic effects of imetelstat. Among subjects with available baseline and post-treatment data, telomerase activity was reduced by >=50% relative to baseline in 23 (51.1%) subjects and 10 (29.4%) subjects in the 9.4 mg / kg treatment group and the 4.7 mg / kg treatment group, respectively, indicating that the PD effect was associated with the anti-tumor activity in the preclinical xenograft in vivo model. In addition, hTERT RNA levels were reduced by >=50% relative to baseline in 35 (61.4%) subjects and 20 (47.7%) subjects in the 9.4 mg / kg treatment group and the 4.7 mg / kg treatment group, respectively (Table 8). A dose-dependent PD effect was thus confirmed, indicating the presence of target engagement.

[0161] Table 8: Reductions from baseline in TA (30%, 50%) or hTERT (50%) achieved by subjects at any time point. Example 4

[0162] Association between PD effect and response at week 24

[0163] A higher proportion of subjects with spleen responders (83.3%) had a reduction in hTERT RNA expression levels of at least >= 50% compared to non-spleen responders (55.6%); a higher proportion of subjects with TSS responses had a reduction in hTERT RNA expression levels of at least >= 50% compared to non-TSS responders (Table 9). hTERT RNA expression levels were measured using whole blood samples collected from patients before and after treatment.

[0164] Table 9: Association between hTERT reduction (50%) from baseline at Week 24 and SVR or TSS response.

[0165] A higher proportion of subjects with a spleen response or TSS response had a decrease in telomerase activity of at least >=30% or >=50% compared to subjects without a spleen response or TSS response (Table 10).

[0166] Table 10: Association between reduction in telomerase activity from baseline at week 24 (30%, 50%) and SVR or TSS response.

[0167] Aspects of the subject matter described above (including embodiments) may be used alone or in combination with one or more aspects or embodiments. Without limiting the content described herein, certain non-limiting content of the present invention is provided below. After reading the present invention, the following content will be apparent to those skilled in the art, and each individually numbered content may be used alone or in combination with any of the preceding or following individually numbered content. This is intended to provide support for all such combinations of the content, and is not limited to the combinations of the content explicitly provided below: 1. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the patient is determined to be in triple negative status, wherein the triple negative status comprises the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. 2. The use according to 1, wherein the myelofibrosis is primary myelofibrosis. 3. The use according to 2, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). 4. The use according to 2, wherein the myelofibrosis is post-essential thrombocythaemia myelofibrosis (post-ET MF). 5. The use according to any one of 1-4, wherein the patient has not previously received JAK inhibitor therapy. 6. The use according to any one of 1-4, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 7. The use according to any one of 1-4, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed. 8. The use according to any one of 1-4, wherein the patient has received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. 9. The use according to any one of 1 to 8, wherein the telomerase inhibitor is imetelstat. 10. The use according to 9, wherein the imetelstat is imetelstat sodium. 11. The method of claim 10, wherein the telomerase inhibitor is imetelstat, and the telomerase inhibitor is administered for 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 cycles, each cycle comprising: About 7-10 mg / kg of imetelstat is given intravenously once every three weeks; About 7-10 mg / kg of imetelstat is given intravenously once a week for three weeks; About 2.5 to 10 mg / kg of imetelstat given intravenously every 3 weeks; or Approximately 0.5-9.4 mg / kg of imetelstat is administered intravenously once every three weeks. 12. The use according to 11, wherein each dosing cycle comprises intravenous administration of about 7-10 mg / kg imetelstat once every three weeks. 13. The use according to 12, wherein each dosing cycle comprises intravenous administration of about 9.4 mg / kg imetelstat once every three weeks. 14. The use according to any one of 1-13, wherein the average relative telomere length is determined by analyzing the relative length of telomere nucleic acids in target cells present in a biological sample of the patient. 15. The use according to any one of 1-14, further comprising selecting patients whose average relative telomere length in target cells present in the corresponding biological sample is determined to be the 50th percentile or lower of the relative telomere length range (determined according to one or more known standards). 16. The use according to any one of 1-15, further comprising screening a patient to determine whether the patient has a high molecular risk (HMR), wherein the HMR comprises 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 1-16, further comprising evaluating the hTERT expression level in a biological sample obtained from the patient after administering the telomerase inhibitor. 18. The use according to 17, wherein the hTERT expression level is reduced by 50% or more relative to the baseline hTERT expression level before administration of the telomerase inhibitor. 19. The use according to any one of 17-18, further comprising changing the dosage, administration frequency, or treatment course of the telomerase inhibitor administered to the subject. 20. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the patient is determined to be at high molecular risk (HMR), wherein a mutation is present in a gene with an HMR comprising at least one selected from the group consisting of additional sex comb-like protein 1 (ASXL1), enhancer of zeste homolog 2 (EZH2), serine and arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2). 21. The use according to 20, wherein the myelofibrosis is primary myelofibrosis. 22. The use according to 21, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). 23. The use according to 21, wherein the myelofibrosis is post-essential thrombocythaemia myelofibrosis (post-ET MF). 24. The use according to any one of 20-23, wherein the patient has not previously received JAK inhibitor therapy. 25. The use according to any one of 20-23, wherein the patient has received JAK inhibitor therapy and the patient is resistant to the JAK inhibitor therapy. 26. The use according to any one of 20-23, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed. 27. The use according to any one of 20-23, wherein the patient has received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. 28. The use according to any one of 20-27, wherein the telomerase inhibitor is imetelstat. 29. The use according to 28, wherein the imetelstat is imetelstat sodium. 30. The method of claim 28, wherein the telomerase inhibitor is imetelstat, and 1, 2, 3, 4, 5, 6, 7, 8 or more dosing cycles are administered, each cycle comprising: About 7-10 mg / kg of imetelstat is given intravenously once every three weeks; About 7-10 mg / kg of imetelstat is given intravenously once a week for three weeks; About 2.5 to 10 mg / kg of imetelstat given intravenously every 3 weeks; or Approximately 0.5-9.4 mg / kg of imetelstat is administered intravenously once every three weeks. 31. The use according to 30, wherein each dosing cycle comprises intravenous administration of about 7-10 mg / kg imetelstat once every three weeks. 32. The use according to 31, wherein each dosing cycle comprises intravenous administration of about 9.4 mg / kg imetelstat once every three weeks. 33. The use according to any one of 20-32, further comprising determining the average relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present in the biological sample of the patient. 34. The use according to any one of 20-33, further comprising selecting patients whose average relative telomere length in target cells present in the corresponding biological sample is determined to be the 50th percentile or lower of the relative telomere length range (determined according to one or more known standards). 35. The use according to any one of 20-34, further comprising screening the patient to determine whether the patient is in a triple negative state, wherein the triple negative state comprises 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 20-35, further comprising evaluating the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor. 37. The use according to 36, wherein the hTERT expression level is reduced by 50% or more relative to the baseline hTERT expression level before administration of the telomerase inhibitor. 38. The use according to any one of 36-37, further comprising changing the dosage, dosing frequency, or treatment course of the telomerase inhibitor administered to the subject. 39. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein the average relative telomere length of cells present in a biological sample of the patient is determined to be at or below the 50th percentile of a range of relative telomere lengths (determined according to one or more known standards). 40. The use according to 39, wherein the myelofibrosis is primary myelofibrosis. 41. The use according to 40, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). 42. The use according to 40, wherein the myelofibrosis is post-essential thrombocythaemia myelofibrosis (post-ET MF). 43. The use according to any one of 39-42, wherein the patient has not previously received JAK inhibitor therapy. 44. The use according to any one of 39-42, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 45. The use according to any one of 39-42, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed. 46. ​​The use according to any one of claims 39-42, wherein the patient has received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. 47. The use according to any one of 39-46, wherein the telomerase inhibitor is imetelstat. 48. The use according to 47, wherein the imetelstat is imetelstat sodium. 49. The method of claim 47, wherein the telomerase inhibitor is imetelstat and is administered for 1, 2, 3, 4, 5, 6, 7, 8 or more dosing cycles, each cycle comprising: About 7-10 mg / kg of imetelstat is given intravenously once every three weeks; About 7-10 mg / kg of imetelstat is given intravenously once a week for three weeks; About 2.5 to 10 mg / kg of imetelstat given intravenously every 3 weeks; or Approximately 0.5-9.4 mg / kg of imetelstat is administered intravenously once every three weeks. 50. The use according to 49, wherein each dosing cycle comprises intravenous administration of about 7-10 mg / kg imetelstat once every three weeks. 51. The use according to 50, wherein each dosing cycle comprises intravenous administration of about 9.4 mg / kg imetelstat once every three weeks. 52. The use according to any one of 39-51, further comprising determining the average relative telomere length by analyzing the relative length of telomere nucleic acids in the cells present in the biological sample of the patient. 53. The use according to any one of 39-52, further comprising evaluating the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor. 54. The use according to 53, wherein the hTERT expression level is reduced by 50% or more relative to the baseline hTERT expression level before administration of the telomerase inhibitor. 55. The use according to any one of 53-54, further comprising changing the dosage, dosing frequency, or treatment course of the telomerase inhibitor administered to the subject. 56. A method of selecting patients most likely to benefit from treatment with a telomerase inhibitor, comprising: Testing the patient for triple negative status, wherein the triple negative status comprises the absence of mutations in each of the JAK2, CALR, and MPL genes; and selecting said patient in triple negative status, The selected patients are most likely to benefit from treatment with a telomerase inhibitor. 57. The method of 56, wherein the patient suffers from myelofibrosis. 58. The method of claim 57, wherein the myelofibrosis is primary myelofibrosis. 59. The method according to 57, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). 60. The method according to 57, wherein the myelofibrosis is post-essential thrombocythaemia myelofibrosis (post-ET MF). 61. The method of any one of 56 to 60, wherein the patient has not previously received JAK inhibitor therapy. 62. The method according to any one of 56 to 60, wherein the patient: Have received JAK inhibitor therapy previously; Have failed prior JAK inhibitor therapy; or Patients who have received prior JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 63. A method according to any one of 56 to 60, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 64. A method according to any one of 56 to 60, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed. 65. The method according to any one of 56 to 60, wherein the patient has received JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. 66. The method of any one of 56 to 65, further comprising administering a telomerase inhibitor to the patient. 67. The method of claim 66, wherein the telomerase inhibitor is imetelstat. 68. The method according to 67, wherein the imetelstat is imetelstat sodium. 69. The method according to any one of 56-68 further comprises obtaining a sample (comprising DNA) from the patient. 70. The method of claim 69, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 71. The method according to 70, wherein the step of obtaining a sample from the patient comprises: Obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof; and DNA is isolated from the bone marrow sample, the peripheral blood sample, or a combination thereof. 72. The method according to 70, wherein the step of obtaining a sample from the patient comprises: obtaining a bone marrow sample from the patient; isolating cells from the bone marrow sample; and DNA is extracted from the isolated cells. 73. The method according to 70, wherein the step of obtaining a sample from the patient comprises: obtaining a peripheral blood sample from the patient; isolating cells from the peripheral blood sample; and DNA is extracted from the isolated cells. 74. A method of selecting patients most likely to benefit from treatment with a telomerase inhibitor, comprising: testing a patient to determine whether the patient has HMR, wherein having HMR comprises a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2, and IDH1 / 2; and selecting the patient having HMR, The selected patients are most likely to benefit from treatment with a telomerase inhibitor. 75. The method of 74, wherein the patient suffers from myelofibrosis. 76. The method of claim 75, wherein the myelofibrosis is primary myelofibrosis. 77. The method of claim 75, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). 78. The method according to 75, wherein the myelofibrosis is post-essential thrombocythaemia myelofibrosis (post-ET MF). 79. The method of any one of 74 to 78, wherein the patient has not previously received JAK inhibitor therapy. 80. The method according to any one of 74 to 78, wherein the patient: Have received JAK inhibitor therapy previously; Have failed prior JAK inhibitor therapy; or Patients who have received prior JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 81. The method of any one of 74 to 78, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 82. A method according to any one of 74 to 78, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed. 83. The method of any one of 74 to 78, wherein the patient has been receiving JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. 84. The method of any one of 74 to 83, further comprising administering a telomerase inhibitor to the patient. 85. The method of 84, wherein the telomerase inhibitor is imetelstat. 86. The method according to 85, wherein the imetelstat is imetelstat sodium. 87. The method according to any one of 74-86 further comprises obtaining a sample (comprising DNA) from the patient. 88. The method of claim 87, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 89. The method of 88, wherein the step of obtaining a sample from the patient comprises: Obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof; and DNA is isolated from the bone marrow sample, the peripheral blood sample, or a combination thereof. 90. The method according to 88, wherein the step of obtaining a sample from the patient comprises: obtaining a bone marrow sample from the patient; isolating cells from the bone marrow sample; and DNA is extracted from the isolated cells. 91. The method of claim 88, wherein the step of obtaining a sample from the patient comprises: obtaining a peripheral blood sample from the patient; isolating cells from the peripheral blood sample; and DNA is extracted from the isolated cells. 92. A method of selecting a patient most likely to benefit from treatment with a telomerase inhibitor, the method comprising: testing a patient for an average relative telomere length by analyzing said relative length of telomere nucleic acids in target cells present in a biological sample of said patient; and Selecting said patient whose average relative telomere length in target cells present in the corresponding biological sample is determined to be at or below the 50th percentile of a relative telomere length range (determined according to one or more known criteria), wherein said selected patient is most likely to benefit from treatment with a telomerase inhibitor. 93. The method of 92, wherein the patient has myelofibrosis. 94. The method of claim 93, wherein the myelofibrosis is primary myelofibrosis. 95. The method of claim 93, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF). 96. The method according to 93, wherein the myelofibrosis is post-essential thrombocythaemia myelofibrosis (post-ET MF). 97. A method according to any one of 92 to 96, wherein the patient has not previously received JAK inhibitor therapy. 98. The method according to any one of 92 to 96, wherein the patient: Have received JAK inhibitor therapy previously; Have failed prior JAK inhibitor therapy; or Patients who have received prior JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 99. A method according to any one of 92 to 96, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy. 100. A method according to any one of 92 to 96, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed. 101. The method of any one of claims 92 to 96, wherein the patient has been receiving JAK inhibitor therapy and has now stopped receiving JAK inhibitor therapy due to treatment-related toxicity or intolerance. 102. The method of any one of 92 to 101, further comprising administering a telomerase inhibitor to the patient. 103. The method of 102, wherein the telomerase inhibitor is imetelstat. 104. The method according to 103, wherein the imetelstat is imetelstat sodium. 105. The method according to any one of 92-104 further comprises obtaining a sample (comprising DNA) from the patient. 106. The method of 105, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof. 107. The method according to 106, wherein the step of obtaining a sample from the patient comprises: Obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof; and DNA is isolated from the bone marrow sample, the peripheral blood sample, or a combination thereof. 108. The method according to 106, wherein the step of obtaining a sample from the patient comprises: obtaining a bone marrow sample from the patient; isolating cells from the bone marrow sample; and DNA is extracted from the isolated cells. 109. The method according to 106, wherein the step of obtaining a sample from the patient comprises: obtaining a peripheral blood sample from the patient; isolating cells from the peripheral blood sample; and DNA is extracted from the isolated cells. 110. A method of monitoring the efficacy of a treatment in a subject with myelofibrosis (MF), the method comprising: measuring the level of hTERT expression in a biological sample obtained from the patient after administration of a telomerase inhibitor; and comparing the hTERT expression level in the biological sample to a baseline hTERT expression level prior to administration of the telomerase inhibitor; Wherein a 50% or greater reduction in the level of hTERT expression in the biological sample identifies a subject with an increased likelihood of benefiting from treatment with the telomerase inhibitor. 111. The method according to 110, wherein the measured or assessed hTERT expression level is hTERT RNA expression level. 112. A method for identifying a patient with myelofibrosis (MF) suitable for treatment with a telomerase inhibitor, the method comprising: measuring the level of hTERT expression in a biological sample obtained from the patient after administration of a telomerase inhibitor; and comparing the hTERT expression level in the biological sample to a baseline hTERT expression level prior to administration of the telomerase inhibitor; The reduction in the hTERT expression level in the biological sample can identify patients with an increased likelihood of benefiting from treatment with the telomerase inhibitor. 113. The method of 112, wherein the hTERT expression level is reduced by 50% or more. Although specific embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, in light of the teachings of the present invention, it will be apparent that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0168] Therefore, the foregoing only illustrates the principles of the present invention. Various structures can be designed, although not explicitly stated or shown here, these designs reflect the principles of the present invention and do not exceed the spirit and scope of the present invention. In addition, all examples and conditional language listed herein are mainly to help readers understand the principles of the present invention and the conception provided by the inventor to further expand the art, and should be interpreted as not being limited by these specific examples and conditions. Moreover, all statements citing the principles, aspects and embodiments of the present invention and their specific examples herein are intended to cover their equivalents in structure and function. In addition, the equivalents are intended to include currently known equivalents and equivalents to be developed in the future, that is, any functionally identical elements developed without considering their structure. Therefore, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. On the contrary, the scope and spirit of the present invention are embodied by the appended claims.

Claims

1. Use of a telomerase inhibitor in the preparation of a medicament for treating patients with myelofibrosis, in: (a) the patient is determined to have a high molecular risk (HMR), wherein the HMR comprises a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, IDH1 and IDH2; and / or (b) the patient suffers from myelofibrosis, wherein the average relative telomere length of cells present in the biological sample of the patient is determined to be at or below the 50th percentile of a range of relative telomere lengths (determined according to one or more known standards); Wherein, said patients determined to have HMR and / or said patients determined to have an average relative telomere length at or below the 50th percentile of a relative telomere length range (determined according to one or more known criteria) have an increased likelihood of benefiting from treatment with said telomerase inhibitor.

2. The use according to claim 1, wherein the patient is determined to have a high molecular risk (HMR), wherein the HMR comprises a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, IDH1 and IDH2.

3. The use according to claim 2, in, The patient is determined to have mutations in at least two genes selected from ASXL1, EZH2, SRSF2, IDH1 and IDH2.

4. The use according to claim 1, wherein the average relative telomere length of cells present in the biological sample of the patient is determined as the 50th percentile or lower of the relative telomere length range (determined according to one or more known standards).

5. The use according to claim 1, wherein the myelofibrosis is selected from the group consisting of primary myelofibrosis, post-polycythemia vera myelofibrosis (post-PV MF), post-essential thrombocythemia myelofibrosis (post-ET MF).

6. The use according to claim 1, wherein the patient has not previously received JAK inhibitor therapy.

7. The use according to claim 1, wherein the patient: The patient has received JAK inhibitor therapy and is resistant to JAK inhibitor therapy; Have received JAK inhibitor therapy and have relapsed; or Patients who have received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

8. The use according to claim 1, wherein the telomerase inhibitor is imetelstat or imetelstat sodium.

9. The use according to claim 1, in, The patient is administered the telomerase inhibitor if the patient is determined to have HMR.

10. The use according to claim 1, in, The telomerase inhibitor is administered to the patient when the patient's average relative telomere length is determined to be at or below the 50th percentile of a range of relative telomere lengths (determined according to one or more known standards).

Images