Methods for identifying patients who may benefit from telomerase inhibitor therapy

Abstract

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 absence of a mutation in each of JAK2, CALR, and MPL; and / or a 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. The patient may have myelofibrosis. The present invention also provides methods for treating myelofibrosis, comprising identifying such a patient.

Description

[0001] Cross-referencing of related patent applications

[0002] Pursuant to Section 119(e) of Chapter 35 of the United States Code, this patent application claims priority over the filing dates of U.S. Provisional Patent Application No. 62 / 712,841, filed July 31, 2018, and U.S. Provisional Patent Application No. 62 / 772,849, filed November 29, 2018, the contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This patent application includes a sequence list, which is submitted electronically in ASCII format, and the contents of which are incorporated herein by reference in their entirety. The ASCII copy is named Sequence_Listing.txt and is 356KB in size. Technical Field

[0005] This patent application relates to a method for identifying patients most likely to benefit from telomerase inhibitor treatment by assessing that each of JAK2, CALR, and MPL is free of mutations; and / or has a high molecular risk (HMR) based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. The invention also relates to a method for treating myelofibrosis in subjects (i.e., patients) with telomerase inhibitors.

[0006] introduce

[0007] Myelofibrosis (MF) is a classic BCR-ABL1-negative chronic myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation and dysregulation of kinase signaling. Cervantes, Hematology, 124(17):2635-2642 (2014). It is also characterized by cytopenia, systemic symptoms, splenomegaly, and the potential to transform into acute myeloid leukemia. Kuykendall et al., Annals of Hematology, 97:435-431 (2018). MF is a poorly prognostic Philadelphia chromosome-negative myeloproliferative neoplasm, in which the JAK1 / JAK2 inhibitor ruxolitinib is currently approved for the treatment of the disease. Ruxolitinib, a Janus kinase (JAK)-1 and JAK-2 inhibitor, is the first-in-class drug approved in the United States for the treatment of intermediate to high myelofibrosis (MF). Pardanani et al., Journal of Blood Cancer; 4(12):e268 (2014). Several other JAK inhibitors are currently under development, 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, and lenalidomide. And thalidomide. Clinical trials are currently underway for MF to evaluate selective JAK inhibitors, histone deacetylase / DNA methyltransferase inhibitors, PI3K inhibitors, target of rapamycin (mTOR) inhibitors in hedgehogs / mammalians, antifibrotic agents, immunomodulators, monoclonal antibodies, and immune checkpoint inhibitors. Shreenivas et al., Emerging Drugs Expert Insights, 23(1):37-49 (2018).

[0008] Other MPNs include essential thrombocythemia (ET) and polycythemia vera (PV). Cervantes (see below). MF can occur de novo (primary MF [PMF]) or after a prior diagnosis of ET or PV (post-ET or post-PVMF). Ibid. According to Cervantes, MF is the clonal proliferation of pluripotent hematopoietic stem cells, in which an abnormal cell population releases various cytokines and growth factors in the bone marrow, causing myelofibrosis and stromal changes. Furthermore, the abnormal cell population can also colonize extramedullary organs such as the spleen and liver. Ibid. Myelofibrosis is 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 older adults, and according to Cervantes, "currently, apart from allogeneic hematopoietic stem cell transplantation (allo-SCT), there are no other radical treatments available for a small number of patients." Ibid.

[0009] In fact, according to Langabeer's research, "most patients with classic myeloproliferative neoplasms (MPNs) (including polycythemia vera, essential thrombocythemia, and essential myelofibrosis) have different disease-driving mutations in their 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 mutations in JAK2 exon 12, MPL exon 10, and CALR exon 9. Ibid.

[0010] According to Spiegel's research, in myelofibrosis (MF), mutations in driver genes such as JAK2, MPL, or CALR affect survival and progression to blast crisis, with triple-negative status (i.e., unmutated JAK2, MPL, and CALR) posing the greatest risk. Spiegel et al., Advances in Hematology, 1(20):1729-1738 (2017). In fact, the absence of JAK2 / MPL / CALR mutations (i.e., triple-negative) is associated with the worst prognosis. Pardanani et al., Journal of Hematologic Cancers; 4(12):e268 (2014); see also Tefferi et al., Hematology, 124(16):2507-13 (2014). Furthermore, mutations in high molecular risk (HMR) genes (e.g., ASXL1, EZH2, IDH1 / 2, and SRSF2) are also associated with poor prognosis. Spiegel et al. Independent of traditional risk factors, the presence of an increasing number of prognostic / “polymeric risk” mutations (i.e., ASXL1, EZH2, SRSF2 and / or IDH-1 / 2 genes) is gradually worsening survival prognosis.

[0011] Guglielmelli et al., Leukemia, 28(9):1804-10 (2014).

[0012] Driver gene mutations in JAK2, MPL, or CALR, whether occurring 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 typical mutations in JAK2, MPL, or CALR have an increased risk of leukemic transformation and shorter OS. Spiegel observed that these mutations were associated with shorter treatment failure time in myelofibrosis patients treated with ruxotinib or molotinib (JAK 1 / 2 inhibitors). Ibid. Similarly, “patients with CALR mutations had significantly lower hemoglobin (mean 8.6 vs 10.7 g / dL; P 5.001) and white blood cell count (mean 11.0 vs 25 g / dL; P 5.033) compared to the clinical characteristics of JAK2-positive, CALR-positive, MPL-positive, and TN MF patients, a trend also reported in other MPN cohorts.” Patel et al., Hematology, 126(6):790-797 (2015). Patel et al. observed a negative correlation between splenic response and time to treatment discontinuation in patients receiving ruxotinib and having ≥3 mutations. Driver gene mutations or triple-negative (JAK2, MPL, CALR) status were observed in myelofibrosis patients who discontinued JAK inhibitor therapy. See, for example, Kuykendall et al. Summary of the Invention

[0013] This invention provides a method for identifying or selecting patients most likely to benefit from telomerase inhibitor (e.g., emexeter) treatment by performing the following tests on patients: no 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: extra comb-like protein 1.

[0014] (ASXL1), Zeste gene enhancer homolog 2 (EZH2), serine and arginine enriched splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2). The patients requiring treatment may have myelofibrosis. This invention also provides a method for treating myelofibrosis in patients requiring such treatment, comprising the step of identifying such patients.

[0015] One embodiment of the present invention is a method for identifying myelofibrosis patients most likely to benefit from telomerase inhibitor therapy, comprising: (a) testing the patient for: (i) a triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) selecting said patients who meet the following criteria: (i) a triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) 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 said selected patients are most likely to benefit from telomerase inhibitor therapy.

[0016] An alternative embodiment of the invention is a method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising: (a) testing the patient for a triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and (b) selecting said patients who meet the following criteria: are triple-negative, wherein said selected patients are most likely to benefit from telomerase inhibitor treatment. An alternative embodiment of the invention is a method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising: (a) performing a high molecular risk (HMR) test on said patients based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and (b) selecting patients 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 for treating myelofibrosis in patients with corresponding triple-negative and / or HMR outcomes with a telomerase inhibitor (e.g., imexistat).

[0017] Another embodiment of the invention is a method for identifying myelofibrosis patients most likely to benefit from telomerase inhibitor treatment, comprising: (a) obtaining a DNA sample from a patient; (b) testing the DNA sample derived from such patients for: (i) a triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) 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 (c) selecting patients who meet the following criteria: (i) are triple-negative, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or (ii) 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, wherein the selected patients are most likely to benefit from telomerase inhibitor treatment. In some embodiments of the method, the DNA sample is obtained from bone marrow, peripheral blood, or both.

[0018] The DNA sample can be obtained by first acquiring a bone marrow sample, a peripheral blood sample, or both, and then isolating the DNA from the bone marrow sample, peripheral blood sample, or both. In one embodiment, the step of obtaining a DNA sample from a patient includes: acquiring a bone marrow sample from the patient; isolating cells from the bone marrow sample; and extracting DNA from the isolated cells. In another embodiment, the step of obtaining a DNA sample from a patient includes: acquiring a peripheral blood sample from the patient; isolating cells (e.g., granulocytes) from the peripheral blood sample; and extracting DNA from the isolated cells.

[0019] Another embodiment of the present invention is a method for identifying myelofibrosis patients most likely to benefit from telomerase inhibitor therapy, comprising performing the following tests on the patient: (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; wherein the presence of (a), (b), or (c) indicates that the patient is most likely to benefit from telomerase inhibitor therapy.

[0020] 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 vera myelofibrosis (post-ET MF). In some 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 an initial response to treatment). In other embodiments, the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0021] The method may further include the step of administering the telomerase inhibitor once such patients are identified. In some embodiments, the telomerase inhibitor is imelmata or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmata is imelmata sodium.

[0022] When treating patients identified by these methods with emelestat, emelestat is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: intravenous administration of approximately 7-10 mg / kg emelestat every three weeks; intravenous administration of approximately 7-10 mg / kg emelestat once weekly for three weeks; intravenous administration of approximately 2.5-10 mg / kg emelestat every three weeks; or intravenous administration of approximately 0.5-9.4 mg / kg emelestat every three weeks. In one embodiment, each dosing cycle comprises intravenous administration of approximately 7-10 mg / kg emelestat every three weeks. In another embodiment, each dosing cycle comprises intravenous administration of approximately 9.4 mg / kg emelestat every three weeks.

[0023] When treating patients identified by these methods with emelestat sodium, emelestat sodium is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: intravenous administration of approximately 7-10 mg / kg emelestat sodium every three weeks; intravenous administration of approximately 7-10 mg / kg emelestat sodium once weekly for three weeks; intravenous administration of approximately 2.5-10 mg / kg emelestat sodium every three weeks; or intravenous administration of approximately 0.5-9.4 mg / kg emelestat sodium every three weeks. In one embodiment, each dosing cycle comprises intravenous administration of approximately 7-10 mg / kg emelestat sodium every three weeks. In another embodiment, each dosing cycle comprises intravenous administration of approximately 9.4 mg / kg emelestat sodium every three weeks.

[0024] Another embodiment of the present invention is a method for treating patients with myelofibrosis using a telomerase inhibitor (e.g., imerestat or imerestat sodium), comprising:

[0025] (i) Screening of the 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 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

[0026] (ii) If such patients are triple-negative and / or at high molecular risk (HMR), the telomerase inhibitor is administered to the patients, wherein triple-negative status is determined based on the absence of mutations in any of JAK2, CALR, and MPL, and 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 can be: primary myelofibrosis, post-PV MF, or post-ET MF. In some embodiments, the patients have not previously received JAK inhibitor therapy. In other embodiments, the patients have previously received JAK inhibitor therapy and have experienced failed JAK inhibitor therapy, or have previously received JAK inhibitor therapy and have now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0027] In some embodiments of the treatment method, the telomerase inhibitor is imemenol, and is administered in 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: intravenous administration of approximately 7-10 mg / kg imemenol every three weeks; intravenous administration of approximately 7-10 mg / kg imemenol once weekly for three weeks; intravenous administration of approximately 2.5-10 mg / kg imemenol every three weeks; or intravenous administration of approximately 0.5-9.4 mg / kg imemenol every three weeks. In some embodiments, each dosing cycle comprises intravenous administration of approximately 7-10 mg / kg imemenol every three weeks. In other embodiments, each dosing cycle comprises intravenous administration of approximately 9.4 mg / kg imemenol every three weeks.

[0028] In some embodiments of a method for identifying or selecting patients most likely to benefit from telomerase inhibitor treatment, the method further comprises determining the mean relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present within the patient's biological sample. In some embodiments of a method for identifying or selecting patients most likely to benefit from telomerase inhibitor treatment, the method further comprises selecting patients whose mean relative telomere length in target cells present within 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). In some embodiments, the telomerase inhibitor is imemenol or a pharmaceutically acceptable salt thereof. In other embodiments, the imemenol is imemenol sodium.

[0029] This invention provides a method for treating patients with myelofibrosis using a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the patient is triple-negative (determined based on the absence of mutations in each of JAK2, CALR, and MPL). In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0030] This invention provides a method for treating patients with myelofibrosis using a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the patient is triple-negative and / or at 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.

[0031] This invention provides a method for treating patients with myelofibrosis using a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the patient has one or more of the following characteristics:

[0032] (a) The mean 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 the range of relative telomere lengths (determined according to one or more known criteria);

[0033] (b) Triple-negative status, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and

[0034] (c) High molecular weight risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0035] The present invention provides a method for identifying myelofibrosis (MF) subjects suitable for treatment with telomerase inhibitors, the method comprising: measuring the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor; and comparing the hTERT expression level in the biological sample with a baseline hTERT expression level prior to administration of the telomerase inhibitor; wherein a decrease in the hTERT expression level in the biological sample may identify patients with an increased likelihood of benefiting from the telomerase inhibitor treatment.

[0036] This invention provides a method for treating myelofibrosis (MF), the method comprising: administering an effective amount of a telomerase inhibitor to a subject with appropriate need; and assessing the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor. In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0037] This invention provides a method for monitoring the therapeutic effect in subjects 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 to a baseline hTERT expression level prior to administration of the telomerase inhibitor; wherein a 50% or greater reduction in the hTERT expression level in the biological sample identifies subjects with an increased likelihood of benefiting from treatment with the telomerase inhibitor. In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0038] The present invention provides a method for selecting patients most likely to benefit from telomerase inhibitor treatment, comprising: testing the patient’s mean relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present in a biological sample of the patient; and selecting patients whose mean 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 the selected patients are most likely to benefit from telomerase inhibitor treatment.

[0039] The present invention provides a method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising: obtaining a biological sample from a patient; determining a mean 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 patients whose mean relative telomere length in the target cells present in the biological sample is determined to be at or below the 50th percentile of a range of relative telomere lengths (determined according to one or more known criteria), wherein the identified patients are most likely to benefit from telomerase inhibitor treatment.

[0040] This invention provides a method for treating patients with myelofibrosis using a telomerase inhibitor, comprising: administering the telomerase inhibitor to the patient if the mean relative telomere length in target cells present in a biological sample of such patients is determined to be at or below the 50th percentile of a relative telomere length range (determined according to one or more known criteria). In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0041] This invention provides a method for monitoring the therapeutic effect in subjects 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 to a baseline hTERT expression level prior to administration of the telomerase inhibitor; wherein a 50% or greater reduction in the hTERT expression level in the biological sample identifies subjects with an increased likelihood of benefiting from treatment with the telomerase inhibitor. In some embodiments, the measured or assessed hTERT expression level is the hTERT RNA expression level. In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0042] The present invention provides a method for identifying myelofibrosis (MF) patients suitable for treatment with telomerase inhibitors, the method comprising: measuring the hTERT expression level in a biological sample obtained from the patient after administration of the telomerase inhibitor; and comparing the hTERT expression level in the biological sample with a baseline hTERT expression level prior to administration of the telomerase inhibitor; wherein a decrease in the hTERT expression level in the biological sample may identify patients with an increased likelihood of benefiting from treatment with the telomerase inhibitor.

[0043] This invention provides a method for monitoring the effect of treatment in subjects with myelofibrosis (MF), the method comprising: measuring telomerase activity levels in a biological sample obtained from the patient after administration of a telomerase inhibitor; and comparing the telomerase activity levels in the biological sample to a baseline telomerase activity level prior to administration of the telomerase inhibitor; wherein a 50% or greater reduction in telomerase activity levels in the biological sample identifies subjects with an increased likelihood of benefiting from treatment with the telomerase inhibitor. In some embodiments, the telomerase inhibitor is imelmeta or a pharmaceutically acceptable salt thereof. In other embodiments, the imelmeta is imelmeta sodium.

[0044] Attached Figure

[0045] A thorough understanding of the foregoing description and the following detailed description of the invention can be obtained by reading the accompanying drawings. The drawings illustrate embodiments of the invention for the purpose of explaining the invention. However, it should be understood that the invention is not limited to the precise devices, examples, and apparatuses shown.

[0046] Figure 1 A waterfall plot of spleen volume reduction (SVR) at week 24 is shown in Example 1 for the 4.7 mg / kg and 9.4 mg / kg treatment groups. SVR is expressed as a percentage change relative to baseline.

[0047] Figure 2 The waterfall plot shows the total symptom score reduction (TSS) in the 4.7 mg / kg and 9.4 mg / kg treatment groups at week 24 in Example 1. TSS is expressed as a percentage change relative to baseline.

[0048] Figure 3 Kaplan-Meier plots of overall survival are shown in the 4.7 mg / kg treatment group, grouped according to the mutation status of the JAK2 / MPL / CALR genes (TN vs. non-TN (MUT)). Specifically, Figure 3 The graph shows the relationship between survival probability and time for patients in triple-negative (TN) status and patients with at least one mutation (MUT).

[0049] Figure 4 Kaplan-Meier plots of overall survival are shown in the 9.4 mg / kg treatment group, grouped according to the mutation status of the JAK2 / MPL / CALR genes (TN vs. non-TN (MUT)). Specifically, Figure 4 The graph shows the relationship between survival probability and time for patients in the 9.4 mg / kg treatment group who were triple-negative (TN) and had at least one mutation (MUT).

[0050] Figure 5Kaplan-Meier plots show the changes in overall survival over time in the 9.4 mg / kg and 4.7 mg / kg treatment groups, grouped according to patient condition.

[0051] Figure 6 Kaplan-Meier plots are shown for overall survival (OS) in the 9.4 mg / kg treatment group, grouped according to the mutation status (TN vs. non-TN) of the JAK2 / MPL / CALR genes. Specifically, Figure 6 The graph shows the relationship between survival probability and time for patients in the 9.4 mg / kg treatment group who were triple-negative (TN) and those with at least one mutation (non-TN).

[0052] Figure 7 Kaplan-Meier plots are shown for overall survival (OS) in the 4.7 mg / kg treatment group, grouped according to the mutation status (TN vs. non-TN) of the JAK2 / MPL / CALR genes. Specifically, Figure 7 The graph shows the relationship between survival probability and time for patients in the 4.7 mg / kg treatment group who were triple-negative (TN) and those with at least one mutation (non-TN).

[0053] Implementation

[0054] This patent application is based on the following findings: the patient has myelofibrosis; the patient's corresponding outcome is triple negative (i.e., no mutations in any of JAK2, CALR, and MPL); and / or the patient belongs to the high molecular risk (HMR) category (determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2) and is able to benefit from treatment with telomerase inhibitors (e.g., emelexistat or emelexistat sodium). Patients with mutations in the ASXL1, EZH1, IDH1 / 2, and SRSF2 genes have an increased risk of early death or leukemic transformation. These patients typically do not benefit from treatment with conventional therapies (e.g., JAK inhibitors). Gisslinger et al., Hematology, 128:1931 (2016). Therefore, the fact that these patients benefit from telomerase inhibitor treatment is unexpected and surprising.

[0055] Accordingly, this patent application provides a method for identifying patients most likely to benefit from treatment with a telomerase inhibitor (e.g., emexeter). The method comprises testing or identifying a patient to determine whether the patient is: triple-negative, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or at 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. This patent application also provides a method for treating myelofibrosis with a telomerase inhibitor (e.g., emexeter), involving identifying patients who meet the following criteria: are triple-negative, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or at 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. Such patients are most likely to benefit from telomerase inhibitor treatment. The telomerase inhibitor (e.g., emexeter) is then administered to the patient. In order to clearly illustrate the invention and not limit it, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments or applications of the invention.

[0056] A. Definition

[0057] Mutations in extra-comb-like protein 1 (ASXL1), Zeste gene enhancer homolog 2 (EZH2), serine and arginine enriched splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2) used in this article should include any mutations in these genes that affect survival and disease progression in patients with myelofibrosis. Furthermore, IDH1 / 2 used in this article should include both IDH1 and IHD2. For exemplary mutations, please refer to the following publications, the contents of which are incorporated herein by reference to gene mutations associated with myelofibrosis: Langabeer, JAK-STAT, 5:e1248011 (2016); Cervantes, Hematology, 124(17):2635-2642 (2014); Patel et al., Hematology, 126(6):790-797 (2015); Spiegel et al., Advances in Hematology Research, 1(20):1729-1738 (2017); Newburry et al., Hematology, 130(9):1125-1131 (2017); Kuykendall et al., Annals of Hematology, 97:435-431 (2018). Exemplary sequences are shown below: Polymer risk (HMR) can be determined based on the presence of mutations in at least one of the following genes: the ASXL1 gene having a nucleic acid sequence such as sequence number 5; the EZH2 gene having a nucleic acid sequence such as sequence number 6; the SRSF2 gene having a nucleic acid sequence such as sequence number 7; the IDH1 gene having a nucleic acid sequence such as sequence number 8; the IDH2 gene having a nucleic acid sequence such as sequence number 9; and combinations thereof.

[0058] 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, and Q780. Mutations in the following numbers: W796, V807, T822, K825, S846, D855, C856, L857, L885, L890, S903, S970, Y974, R965, G967, V962, L992, S1028, Q1039, R1073, E1102, H1153, S1209, S1231, A1312, F1305, P1377, R1415, and I1436. In some embodiments, the mutations are Q575X, Q588X, Y591X, Y591N, Q592X, S604F, L614F, Q623X, A627G, E635R, T638V, A640G, G646W, G658X, R678K, C687R, C687V, D690G, R693X, Y700X, G704R, G704W, E705X, Q708X, G710E, L721C, E727X, V751L, P763R, Q780X, and W7. 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.

[0059] In some embodiments, the target mutation in the EZH2 gene includes mutations in W60, R63, P312, F145, N182, R288, Q328, Q553, R566, T573, R591, R659, D677, V679, R690, A702, V704, E726, D730, and / or Y733. In some embodiments, the mutation is a W60X mutation, an R63X mutation, a P312S mutation, an F145S mutation, an N182D mutation, an R288Q mutation, a Q328X mutation, a Q553X mutation, an R566H mutation, a T573I mutation, an R591H mutation, an R659K mutation, a D677H mutation, a V679M mutation, an R690H mutation, an A702V mutation, a V704L mutation, an E726V mutation, a D730X mutation, and / or a Y733X mutation.

[0060] In some embodiments, the target mutation in the SRSF2 gene includes the P95 mutation. In some embodiments, the mutation is a P95H mutation, a P95L mutation, or a P95R mutation.

[0061] In some embodiments, the target mutation in the IDH1 / 2 gene includes R132 and / or R140 mutations. In some embodiments, the mutation is an R132G mutation, an R132H mutation, or an R140Q mutation.

[0062] In some embodiments, the targeted mutation includes the mutations listed below:

[0063]

[0064]

[0065] As used in this article, "triple-negative status," "triple-negative," or "TN" refers to the test results of patients who do not have mutations in any of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. Triple-negative status can be determined based on the absence of mutations in any of the following genes: the JAK2 gene with a nucleotide sequence such as sequence number 2; the CALR gene with a nucleotide sequence such as sequence number 3; and the MPL gene with a nucleotide sequence such as sequence number 4.

[0066] In some embodiments, a triple-negative state includes the absence of mutations in the JAK2 gene, such as G335, F556, G571, V617, and / or V625 mutations. For example, a triple-negative state may include the absence of G335D, F556V, G571S, V617F, and / or V625S mutations in the JAK2 gene.

[0067] In some embodiments, a triple-negative state includes the absence of mutations in the MPL gene, for example,

[0068] T119, S204, P222, E230, V285, R321, S505, W515, Y591, and / or R592 mutations. For example, a triple-negative status may include the absence of the following mutations in the MPL gene: T119I, S204F, S204P, P222S, E230G, V285E, R321W, S505N, W515R, W515L, Y591N, and / or R592Q.

[0069] In some embodiments, a triple-negative state includes the absence of mutations in the CALR gene, such as L367, K368, E381, K385, and / or E396 mutations. For example, a triple-negative state may include the absence of L367T, K368R, K385N, E381A, and / or E396del mutations in the CALR gene.

[0070] In some embodiments, the targeted mutation includes the mutations listed below:

[0071] Gene protein change JAK2 p.Val617Phe Misunderstanding JAK2 p.Val617Phe Misunderstanding JAK2 p.Val617Phe Misunderstanding MPL p.Ser505Asn Misunderstanding MPL p.Trp515Lys Misunderstanding MPL p.Trp515Leu Misunderstanding CALR p.Leu367Thrfs*46 Shift code CALR p.Leu367fs Shift code CALR p.Glu381Ala Misunderstanding CALR p.Lys385Asnfs*47 Shift code CALR p.Glu396del Missing within the frame CALR p.Lys368Argfs*51 Shift code

[0072] In this article, "failed" JAK inhibitor therapy means that the disease is resistant to the therapy, or that the patient is resistant to the therapy, or that the disease has relapsed despite an initial response to treatment.

[0073] When referring to measurable values ​​(e.g., quantity, time interval, etc.), the term “about” as used herein means a variation of ±20% to ±0.1% of the specified value, preferably ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, because such variation is suitable for performing the disclosed methods.

[0074] The term "pharmaceutically acceptable salt" refers to an acceptable salt suitable for administration to patients such as mammals (a salt containing an antiion and having 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" also refers to a pharmaceutically acceptable salt of a compound, wherein the salt is derived from various organic and inorganic antiions well known in the art, and includes, for example, sodium; when the molecule contains a basic functional group, it also includes salts of organic or inorganic acids, such as hydrochlorides. Pharmaceutically acceptable salts include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine penicillin, calcium, theophylline, ethylenediamine, lysine, lithium, magnesium, meglumine, procaine, potassium, sodium, tromethamine, N-methylglucosamine, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, zinc, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine salts.

[0075] The term "its salt" 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). Preferably, the salt is a pharmaceutically acceptable salt. For example, salts of the compounds of the present invention include salts 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 the anionic component of the salt. Target salts include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine penicillin, calcium, theophylline, ethylenediamine, lithium, magnesium, meglumine, procaine, N-methylglucosamine, 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 internucleotide bonds), such oligonucleotides may also include any suitable salt form. In some embodiments, for simplicity, the acidic form of the nucleotide-to-nucleotide bond is depicted. In some cases, the salt of the target compound is a monovalent cationic salt. In some cases, the salt of the target compound is a divalent cationic salt. In some cases, the salt of the target compound is a trivalent cationic salt. A "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 both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0076] "Stereoisomers" are compounds that have the same atomic connectivity but different spatial arrangements of atoms. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers. For any group containing one or more substituents disclosed in this invention, it should be understood that the group does not contain any substitution or substitution pattern that is spatially impractical and / or synthetically infeasible. All stereoisomers are intended to be included within the scope of this invention.

[0077] Those skilled in the art will recognize that other tautomer arrangements of the groups described herein are also possible. It should be understood that, unless explicitly stated otherwise, all tautomer forms of the target compound are covered by the structure which describes one possible tautomer arrangement of the groups of the compound.

[0078] It is intended to include solvates of pharmaceutically acceptable salts of stereoisomers of the target compound. These are intended to be included within the scope of this invention.

[0079] Before describing certain embodiments in more detail, it should be understood that the invention is not limited to the described embodiments, as differences will certainly exist in actual implementation. It should also be understood that the terminology used herein is for describing certain embodiments only and is not intended to limit the inventive concept; the scope of the invention will be defined only by the appended claims.

[0080] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within that range, is included within the scope of this invention. Unless the context explicitly 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 range and also within this 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 both of the included limits is also included within this invention.

[0081] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials to those described herein may also be used in the implementation or testing of this invention, representative exemplary methods and materials are described below.

[0082] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and the purpose of their inclusion is to disclose and describe methods and / or materials relating to the cited publications. References to any publication refer to its content disclosed prior to the filing date and should not be construed as an admission that the present invention is not entitled to precede such publication by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may require separate verification.

[0083] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a precondition for the use of specialized terms related to the stated elements of a claim, such as “alone,” “only,” or the use of the limiting word “negative.”

[0084] Each individual embodiment described and listed herein has hierarchical components and features that can be quickly decomposed or combined with features of any of the other embodiments without departing from the scope and spirit of the invention. Any of the stated methods may be implemented in the order of the stated events or in any other logically possible order.

[0085] B. Identify patients most likely to benefit from telomerase inhibitor therapy.

[0086] In one aspect, the present invention provides a method for identifying or selecting patients most likely to benefit from telomerase inhibitor therapy. The method relies on identifying patients who are triple-negative (those without mutations in any of the JAK2, CALR, and MPL genes) or patients with 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. These triple-negative or HMR patients are most likely to benefit from telomerase inhibitor therapy (e.g., imelustat or imelustat sodium).

[0087] The myelofibrosis can be primary myelofibrosis, post-polycythemia vera myelofibrosis (post-PV MF), or post-thrombocythemia vera myelofibrosis (post-ET MF). In some embodiments, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and has experienced 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 an initial response to treatment). In other embodiments, the patient has previously received JAK inhibitor therapy and has now discontinued 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 discontinued JAK inhibitor therapy.

[0088] 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 yet 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 now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0089] In one embodiment, the invention provides a method for selecting patients most likely to benefit from telomerase inhibitor treatment by testing one or more of the following: a triple-negative status, 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 subjected to a high molecular risk (HMR) test, 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 invention provides a method for selecting patients most likely to benefit from telomerase inhibitor treatment by testing: a triple-negative status, 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 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 another embodiment, the present invention provides a method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising performing the following tests on the patient: (a) a triple-negative status, determined based on the absence of any mutations in the JAK2, CALR, and MPL genes; (b) a 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. In this embodiment, the presence of (a), (b), or (c) indicates that the patient is most likely to benefit from telomerase inhibitor treatment.

[0090] Another embodiment of the present invention is a method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising:

[0091] a. Perform the following tests on the patient:

[0092] i. Triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or

[0093] ii. Polymer risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0094] b. Select patients who meet the following criteria:

[0095] i. Triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and / or

[0096] ii. Possesses 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.

[0097] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0098] Another embodiment of the invention is a method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising: testing a patient for triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and selecting said patients who meet the following criteria: are triple-negative, determined based on the absence of mutations in the JAK2, CALR, and MPL genes, wherein said selected patients are most likely to benefit from telomerase inhibitor treatment. In one embodiment, this method further includes performing a polymeric risk (HMR) test on the patient, determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2, and includes selecting said patients with HMR.

[0099] In some embodiments of any of these methods, no mutations have occurred in the coding regions (exons) of the JAK2, CALR, and MPL genes of the triple-negative patient.

[0100] Furthermore, in other embodiments of any of these methods, the polymeric risk (HMR) is determined based on the presence of mutations in the coding regions (exons) of at least one of the ASXL1, EZH2, SRSF2, and IDH1 / 2 genes.

[0101] In some embodiments, polymeric risk (HMR) is determined by detecting mutations in ASXL1, EZH2, SRSF2, or IDH1 / 2, or combinations thereof. In some embodiments, the method includes detecting a mutation in ASXL1. In some embodiments, the method includes detecting a mutation in EZH2. In some embodiments, the method includes detecting a mutation in SRSF2. In some embodiments, the method includes detecting a mutation in IDH1 / 2. In some embodiments, the method includes detecting mutations in both ASXL1 and EZH2. In some embodiments, the method includes detecting mutations in both ASXL1 and SRSF2. In some embodiments, the method includes detecting mutations in both ASXL1 and IDH1 / 2. In some embodiments, the method includes detecting mutations in EZH2 and SRSF2. In some embodiments, the method includes detecting mutations in both EZH2 and IDH1 / 2. In some embodiments, the method includes detecting mutations in both SRSF2 and IDH1 / 2. In some embodiments, the method includes detecting mutations in ASXL1, EZH2, and SRSF2. In some embodiments, the method includes detecting mutations in ASXL1, EZH2, and IDH1 / 2. In some embodiments, the method includes detecting mutations in EZH2, SRSF2, and IDH1 / 2. In some embodiments, the method includes detecting mutations in ASXL1, EZH2, SRSF2, and IDH1 / 2. In another embodiment of the invention, the invention provides a method for identifying or selecting patients in a patient population most likely to benefit from telomerase inhibitor therapy. In this method, patients in the patient population with mutations in each of the corresponding JAK2, CALR, and MPL genes are screened to identify triple-negative patients in the population. In an alternative embodiment, the method relies on identifying triple-negative patients without typical mutations in each of the corresponding JAK2, MPL, and CALR genes.

[0102] In some embodiments, the method further includes the step of obtaining a patient DNA sample. The patient sample may be collected from a DNA sample obtained from bone marrow, peripheral blood, or both. Therefore, in some embodiments, the method of the present invention includes obtaining a patient blood sample and isolating (extracting) DNA from the patient blood sample. The method may also include the step of isolating cells (e.g., granulocytes) from the patient blood sample. Similarly, the method of the present invention includes obtaining a bone marrow sample and isolating (extracting) DNA from the bone marrow sample. The method may also include the step of isolating cells from the patient bone marrow sample.

[0103] The patient's DNA sample is tested using conventional techniques, with or without mutations in each of the JAK2, CALR, and MPL genes. Alternatively, the patient's DNA sample is tested using conventional techniques, with mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2. In some embodiments, the patient's 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.

[0104] In some embodiments, the test of the DNA sample may be a next-generation sequencing assay using the Illumina MiSeq platform disclosed in the following publication: Patel et al., Blood, 126(6):790-797 (2015), the contents of which are incorporated herein by reference to DNA sample testing.

[0105] C. Pharmacodynamics (PD)

[0106] This invention is partly based on pharmacodynamic effects that demonstrate an association between response to telomerase inhibition therapy and a reduction in telomerase hTERT expression levels relative to baseline in subjects with myelofibrosis. In some cases, a higher proportion of subjects who developed a clinical response (splenic or symptomatic) to telomerase inhibition therapy at week 24 had a 50% or greater reduction in hTERT RNA expression levels compared to unresponsive subjects.

[0107] This invention provides stratification and selection of patients who may benefit from telomerase inhibition therapy applicable to myelofibrosis, and provides methods for monitoring the response, relapse, and prognosis of subjects receiving treatment.

[0108] The present invention includes methods for selecting myelofibrosis (MF) subjects for treatment with telomerase inhibitors, and methods for treating MF. The invention also provides methods for monitoring the therapeutic effect in MF subjects. In some cases, the pharmacokinetic effect upon which an embodiment of the target method is based is a reduction in hTERT RNA expression of 50% or more, for example 60% or more, 70% or more, 80% or more, or 90% or more.

[0109] The telomerase ribonucleoprotein is composed of components or subunits, two of which are telomerase RNA template (hTR) and telomerase reverse transcriptase protein (hTERT). hTERT expression levels can be assessed, determined, and / or measured using any suitable method. Various methods can be applied to the amplification, detection, and measurement of telomerase components or related protein mRNAs in body fluids. The target methods and assays applicable to the target method include, but are not limited to, real-time quantitative RT-PCR assays, such as TaqMan-based fluorescence methods, immunohistochemical methods applicable to protein expression, and methods described in the following publications: U.S. Patent No. 6,607,898; Bieche et al., Clinical Cancer Research, February 1, 2000 (6)(2)452-459; Terrin et al. (“Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and delineates patient subgroups 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 splicing variants in chronic lymphocytic leukemia.” Experimental Hematology, 2013, 41:615-626).

[0110] The hTERT expression level can be assessed or measured in any suitable target cells or biological sample. Target cells can be any suitable cells from the patient, including but not limited to cells from the patient's bone marrow or peripheral blood. In some cases, the target cells are isolated from the patient's bone marrow sample. In some cases, the target cells are isolated from the patient's peripheral blood sample. The target cells may be granulocytes.

[0111] hTERT RNA expression levels can be assessed or measured in RNA samples using any suitable method. RNA samples can be obtained by first acquiring a bone marrow sample, a peripheral blood sample, or both, and then isolating the RNA from said bone marrow sample, peripheral blood sample, or both. In one embodiment, the step of obtaining an RNA sample from a patient includes: obtaining a bone marrow sample from the patient; isolating cells from said bone marrow sample; and extracting RNA and / or DNA from said isolated cells. In another embodiment, the step of obtaining an RNA sample from a patient includes: obtaining a peripheral blood sample from the patient; isolating cells (e.g., granulocytes) from said peripheral blood sample; and extracting RNA and / or DNA from said isolated cells.

[0112] D. Treatment

[0113] Aspects of the invention include methods for treating myelofibrosis in subjects (i.e., patients) who meet the following criteria and have a corresponding need: being triple-negative, determined based on the absence of any mutations in the JAK2, CALR, and MPL genes (i.e., no mutations in these genes or no mutations occurring in these genes); and / or having 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. One embodiment of the invention is a method for treating myelofibrosis in subjects (i.e., patients) who meet the following criteria and have a corresponding need: being triple-negative, determined based on the absence of any mutations in the JAK2, CALR, and MPL genes (i.e., no mutations in these genes or no mutations occurring in these genes). In one embodiment, the myelofibrosis is primary myelofibrosis. In another embodiment, the myelofibrosis is post-PV MF. In an alternative embodiment, the myelofibrosis is post-ET MF.

[0114] In some embodiments of the treatment method, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and has 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 an initial response to treatment). In alternative embodiments of the treatment method, the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. In some embodiments, the treatment method further includes prophylactic administration of diphenhydramine (25 to 50 mg) and hydrocortisone (100 to 200 mg) or their equivalents.

[0115] The subject is a mammal that requires 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 used for drug screening, characterization, and evaluation, such as a mouse and a rat), and other mammals. The terms patient, subject, and individual are used interchangeably herein.

[0116] As used herein and well known in the art, "treatment" refers to a method for obtaining beneficial or desired outcomes, including clinical outcomes. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief or improvement of one or more symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), prevention of disease spread, delay or slowing of disease progression, improvement or mitigation of said disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also imply a prolonged survival compared to expected survival without treatment.

[0117] E. Telomerase inhibitors

[0118] The methods of this invention can be used to identify patients most likely to benefit from treatment with any suitable telomerase inhibitor. Furthermore, 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, particularly an oligonucleotide as 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, it may be possible to administer one or more telomerase inhibitors (e.g., two or three telomerase inhibitors) to mammals to treat hematologic malignancies.

[0119] Imestar

[0120] In some embodiments, the telomerase inhibitor is imeresta, comprising its tautomers and its salts, for example, pharmaceutically acceptable salts. imeresta is a first-in-class novel telomerase inhibitor that has demonstrated clinical activity in hematologic malignancies (Baerlocher et al., NEJM 2015, 373:920-928; Tefferi et al., NEJM 2015, 373:908-919) (as shown below):

[0121]

[0122] Wherein “nps” represents the thioaminophosphate bond —NH—P(═O)(SH)—O—, which connects the 3' end carbon of a nucleoside to the 5' end carbon of the adjacent nucleoside.

[0123] In some cases, the telomerase inhibitor is imelistar sodium, including its tautomers. Imelistar sodium is the sodium salt of imelistar, which is a synthetic lipid-conjugated 13-meric oligonucleotide N3'P5'-thioaminophosphate. Imelistar sodium is a telomerase inhibitor, which is a covalently lipidated 13-meric oligonucleotide (shown below) complementary to the template region of human telomerase RNA (hTR). The chemical name of imelistar sodium is: DNA, d(3'-amino-3'-deoxy-P-thio)(TAGGGTTAGACAA), 5'-[O-[2-hydroxy-3-(hexadecylamino)propyl]thiophosphate], sodium salt (1:13) (Serial No.: 1). Imelistar sodium cannot act via an antisense mechanism and therefore does not have the side effects commonly observed in such therapies.

[0124]

[0125] Imesta sodium

[0126] Unless otherwise stated or clearly understood from the context, references to imesta herein also include its tautomers and its salts, such as pharmaceutically acceptable salts. As previously mentioned, imesta sodium is specifically the sodium salt of imesta. Unless otherwise stated or clearly understood from the context, references to imesta sodium herein also include all its tautomers.

[0127] Imesta and imesta sodium can be produced, formulated, or obtained as described elsewhere (see, for example, Asai et al., Cancer Research, 63:3931-3939 (2003); Herbert et al., Oncogenes, 24:5262-5268 (2005); and Gryaznov, Chemistry and Biodiversity, 7:477-493 (2010)). Unless otherwise stated or clearly understood from the context, references to imesta sodium in this document also include its salts. As previously mentioned, imesta sodium is, in particular, the sodium salt of imesta.

[0128] Imesta targets the RNA template of telomerase and inhibits telomerase activity and cell proliferation in various mouse cancer cell lines and tumor xenografts. Phase 1 studies involving patients with breast cancer, non-small cell lung cancer and other solid tumors, multiple myeloma, or chronic lymphocytic leukemia provided information on the pharmacokinetic and pharmacodynamic characteristics of the drug. Subsequent Phase 2 studies involving patients with essential thrombocytosis showed that decreased platelet activity was accompanied by a significant reduction in the allele load of JAK2 V617F and CALR mutants. Imesta sodium is usually administered intravenously; other routes of administration, such as intrathecal, intratumoral, and oral administration, are anticipated when implementing the target method. Imesta sodium can be administered at doses equivalent to those commonly used clinically. In some embodiments, imesta sodium is administered as described elsewhere herein.

[0129] The specific embodiments are based on any of the other embodiments, wherein imesta is limited to imesta sodium.

[0130] F. Pharmaceutical Composition

[0131] For ease of administration, the telomerase inhibitors (e.g., as described herein) can be formulated into various dosage forms for administration purposes. In some cases, the telomerase inhibitors are administered as pharmaceutical compositions. The carrier or diluent of the pharmaceutical composition must be "acceptable" in terms of compatibility with the other components of the composition and harmlessness to the recipient. The pharmaceutical composition may be a single dosage form suitable, in particular, for oral, rectal, transdermal, parenteral, or inhalation administration. In some cases, administration may be by intravenous injection. For example, in preparing the compositions for oral dosage forms, any commonly used pharmaceutical solvent, such as water, ethylene glycol, oil, alcohol, etc., can be used for oral liquid formulations (e.g., suspensions, syrups, elixirs, emulsions, and solutions); and 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 forms of oral dosage units due to their ease of administration, and solid pharmaceutical carriers are obviously used in this case. For parenteral compositions, although they may typically include other components such as those that aid dissolution, the carrier typically (at least predominantly) comprises sterile water. For example, injectable solutions can be prepared in which the carrier comprises an aqueous solution of physiological saline, a glucose solution, or a mixture of physiological saline and glucose solutions. Injectable solutions containing the telomerase inhibitors described herein can be formulated in oils to prolong efficacy. 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 oils and other oils. Injectable suspensions can also be prepared, in which case suitable liquid carriers, suspending agents, etc., can be used. Solid form formulations intended to be converted to a liquid form shortly before use are also included. In compositions suitable for transdermal administration, the carrier optionally comprises a penetration enhancer and / or a suitable wetting agent, optionally combined with a small amount of a suitable additive of any nature, wherein the additive does not produce significant adverse effects on the skin. The additive may facilitate transdermal administration and / or may contribute to the preparation of the desired composition. The composition can be applied in various ways, such as as a transdermal patch, a spot treatment, or an ointment.

[0132] For ease of administration and dosage uniformity, it is particularly advantageous to formulate the above-described pharmaceutical compositions into unit dosage forms. As used herein, a unit dosage form refers to a physically discrete unit suitable as a single dose, each unit containing a predetermined amount of active ingredient calculated based on the amount required to produce the desired therapeutic effect when combined with the necessary drug carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, rice paper capsules, suppositories, injectable solutions or suspensions, and multiple combinations thereof.

[0133] 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-cyclodextrins or their derivatives, especially hydroxyalkyl-substituted cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Similarly, co-solvents (e.g., alcohols) may also improve the solubility and / or stability of the telomerase inhibitors in pharmaceutical compositions.

[0134] Depending on the route of administration, the pharmaceutical composition preferably comprises 0.05 to 99% (by weight), more preferably 0.1 to 70% (by weight), even more preferably 0.1 to 50% (by weight), of the telomerase inhibitor described herein, and 1 to 99.95% (by weight), more preferably 30 to 99.9% (by weight), even more preferably 50 to 99.9% (by weight), of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

[0135] G. Dosing and Dosing Regimen

[0136] The dosing frequency can be any frequency that reduces the severity of myelofibrosis symptoms without causing significant toxicity to the subject. For example, the dosing frequency can be from about once every two months to about once weekly, or from about once monthly to about twice monthly, or about once every six weeks, about once every five weeks, or about once every four weeks, or about once every three weeks, or about once every two weeks, or about once weekly. During treatment, the dosing frequency can remain constant or can be variable. Treatment with a composition containing one or more telomerase inhibitors may include rest periods. For example, a composition containing telomerase inhibitors may be administered weekly for three weeks, followed by a two-week rest period, and such a regimen may be repeated multiple times. As with effective doses, various factors can influence the actual dosing frequency for a particular application. For example, depending on the effective dose, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of myelofibrosis and related symptoms, the dosing frequency may need to be increased or decreased.

[0137] The effective duration of administration of a composition containing a telomerase inhibitor (e.g., ememestat or ememestat sodium) can be any duration during which the severity of myelofibrosis symptoms is reduced (e.g., as described herein) without causing significant toxicity to the subject. Therefore, the effective duration can range from one month to several months or several years (e.g., one month to two years, one month to one year, three months to two years, three months to ten months, or three months to 18 months). Generally, the effective duration of treatment for myelofibrosis can range from two months to twenty months. In some cases, the effective duration can be the survival time of a single subject. Many factors can influence the actual effective duration of a particular treatment. For example, the effective duration can vary with the frequency of administration, the effective dose, the use of multiple therapeutic agents, the route of administration, and the severity of myelofibrosis and related symptoms.

[0138] In some cases, the treatment process 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 has decreased. For example, biopsy techniques can be used to assess the severity of myelofibrosis symptoms (e.g., as described herein).

[0139] The telomerase inhibitors used in the target method can be administered at any therapeutically effective dose, such as doses equivalent to those commonly used clinically. Specific dosing regimens (e.g., recommended effective doses) for known and approved anticancer drugs are known to physicians and are given in publications such as product instructions, which can be obtained from: Physician's Desk Reference, 2003, 57th edition, Medical Economics Company, Inc., Oladell, NJ; Goodman Gilman Therapeutics: Pharmacological Basis, 2001, 10th edition, McGraw-Hill, NY; and / or may be available from the FDA and / or discussed in medical literature.

[0140] In some aspects, the dose of the telomerase inhibitor emelesta sodium administered to the subject is from about 1.0 mg / kg to about 13.0 mg / kg. In other aspects, the dose of the telomerase inhibitor is from about 4.5 mg / kg to about 11.7 mg / kg, or from about 6.0 mg / kg to about 11.7 mg / kg, or from about 6.5 mg / kg to about 11.7 mg / kg. In some embodiments, the dose of the 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, etc. 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 the following: mg / kg, 11.3 mg / kg, 11.4 mg / kg, 11.5 mg / kg, 11.6 mg / kg, 11.7 mg / kg, 11.8 mg / kg, 11.9 mg / kg, 12 mg / kg, 12.1 mg / kg, 12.2 mg / kg, 12.3 mg / kg, 12.4 mg / kg, 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg, or 13 mg / kg.

[0141] In some embodiments, the effective amount of the telomerase inhibitor administered to the individual comprises any one of at least about 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor administered to the individual is any one of about 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 6.5 mg / kg, 7.5 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In various embodiments, the effective amount of telomerase inhibitor administered to the individual includes less than any one of about 350 mg / kg, 300 mg / kg, 250 mg / kg, 200 mg / kg, 150 mg / kg, 100 mg / kg, 50 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 7.5 mg / kg, 6.5 mg / kg, 5 mg / kg, 3.5 mg / kg, 2.5 mg / kg, 1 mg / kg, or 0.5 mg / kg of telomerase inhibitor.

[0142] Exemplary dosing frequencies suitable for pharmaceutical compositions including telomerase inhibitors include, but are not limited to, once daily; every other day; twice weekly; three times weekly; once weekly without interruption; once weekly for three weeks; once every three weeks; once every two weeks; once weekly for two weeks. In some embodiments, the pharmaceutical composition is administered about once weekly, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. In some embodiments, the composition is administered at least about 1x, 2x, 3x, 4x, 5x, 6x, or 7x per week (i.e., daily), or three times daily or twice daily. In some embodiments, the time interval between each dosing 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 dose is greater than any one of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, the dosing schedule does not include any interruption to dosing. In some embodiments, the time interval between each dose does not exceed about one week.

[0143] Telomerase inhibitors such as imeprestat (e.g., imeprestat sodium) can be administered by any suitable method. For example, telomerase inhibitors such as imeprestat (e.g., imeprestat sodium) can be administered intravenously every 4 weeks for a duration of time (e.g., 1, 2, 3, 4, 5 hours). In some embodiments, imeprestat is administered intravenously once weekly at a dose of 7-10 mg / kg for a duration of about 2 hours. In some embodiments, imeprestat is administered intravenously every 3 weeks at a dose of about 0.5-9.4 mg / kg for a duration of about 2 hours. In one embodiment, imeprestat is administered intravenously every 4 weeks at a dose of 0.5-5 mg / kg for a duration of about 2 hours. In one embodiment, imeprestat is administered intravenously every 3 weeks at a dose of about 2.5-10 mg / kg for a duration of about 2 hours. Alternatively, imeprestat is administered intravenously every 4 weeks at a dose of about 0.5-9.4 mg / kg for a duration of about 2 hours.

[0144] In some embodiments of the method, imexert is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: intravenous administration of approximately 7-10 mg / kg imexert every three weeks; intravenous administration of approximately 7-10 mg / kg imexert once weekly for three weeks; intravenous administration of approximately 2.5-10 mg / kg imexert every three weeks; or intravenous administration of approximately 0.5-9.4 mg / kg imexert every three weeks. In some cases, each dosing cycle comprises intravenous administration of approximately 7-10 mg / kg imexert every three weeks. In some cases, each dosing cycle comprises intravenous administration of approximately 9.4 mg / kg imexert approximately every three weeks.

[0145] In one embodiment of the invention, imeresta is administered intravenously at a dose of about 7-10 mg / kg imeresta every three weeks, following prophylactic administration of an antihistamine, corticosteroid, or both. In other embodiments, imeresta is administered intravenously at a dose of about 9.4 mg / kg, or about 7.0 mg / kg to 9.8 mg / kg imeresta, every three weeks, following prophylactic administration of an antihistamine, corticosteroid, or both imeresta.

[0146] In some embodiments, imeresist is administered at a dose of about 7.5 mg / kg, or from about 7.0 mg / kg to 7.7 mg / kg, every three weeks for at least three cycles, followed by an increase in the dose. In some embodiments, the dose of imeresist may be increased to about 9.4 mg / kg, or from about 8.8 mg / kg to about 9.6 mg / kg, provided that the ANC and platelet nadir do not decrease to about 1.5 x 10⁻⁶. 9 / L to approximately 75x 109 The concentration was between 1 / L and no non-hematologic toxicity of grade 3 or higher was observed.

[0147] It should be understood that cancer treatment sometimes involves multiple “rounds” or “cycles” of drug administration, where each cycle includes one or more administrations of the drug according to a specified schedule (e.g., administration for three consecutive days every three weeks; administration once a week; etc.). For example, anticancer drugs may be administered for one to eight cycles or longer. When administering more than one drug (e.g., two drugs) 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) can be coordinated so that two drugs are administered on the same day at least at some point, or that the drugs are administered consecutively for several days at least at some point.

[0148] In some embodiments, the imesta may be administered according to a regimen involving dose reduction. In one embodiment, the patient is initially given a dose of about 9.4 mg / kg every three weeks, then the dose is changed to about 7.5 mg / kg every three weeks, and then the dose is changed to about 6.0 mg / kg every three weeks.

[0149] As is known in the art, if toxicity is observed, or for the convenience of the patient, treatment with the cancer treatment drug may be temporarily suspended without departing from the scope of the invention, and then administration may be resumed.

[0150] Aspects of the method include identifying or selecting patients most likely to benefit from treatment based on the relative telomere length in their target cells (e.g., as described herein). Target cells can be any suitable cells from the patient, including but not limited to cells from the patient's bone marrow or peripheral blood. In some cases, the target cells are isolated from a bone marrow sample from the patient. In some cases, the target cells are isolated from a peripheral blood sample from the patient. The target cells may be granulocytes. In some cases, each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes in the patient is unmutated; and the patient's target cells have a particularly short telomere length. As used herein, short telomere length refers to a telomere length less than or equal to the median or mean telomere length compared to a suitable control (e.g., one or more known standards described herein). Therefore, the target method may further include determining the relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present in the biological sample of the individual; when the average relative telomere length in the target cells present in the biological sample of the individual is determined to be at or below the 50th percentile of the relative telomere length range (determined according to one or more known criteria), for example, at or below the 45th percentile, 40th percentile, 35th percentile, 30th percentile, 25th percentile, 20th percentile, or even lower of the relative telomere length range (determined according to one or more known criteria), individuals who will benefit from telomerase inhibitor treatment are selected.

[0151] In some instances of the method, the one or more known criteria are ranges of telomere length established based on a variety of naturally occurring target cells (e.g., as described herein) from multiple individuals diagnosed with the disease. In some instances of the method, the one or more known criteria are characterized cell lines. A “characterized cell line” means that the relative telomere nucleic acids of the cells in the cell line are known and relatively constant.

[0152] 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 average 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, 40th percentile or lower, 35th percentile or lower, 30th percentile or lower, 25th percentile or lower, 20th percentile or lower, 15th percentile or lower, 10th percentile or lower, or 5th percentile or lower of the relative telomere length range (determined according to one or more known criteria).

[0153] Telomere length in target cells can be determined using any suitable assay, including but not limited to qPCR, telo-FISH, or Southern Blot assays, as described by Bassett et al. in U.S. Patent No. 9,200,327. In one aspect, telomere length can be determined by measuring the average length of the terminal restriction fragment (TRF). The TRF is defined as the length (typically the average length) of the fragment obtained by completely digesting genomic DNA with a restriction enzyme that does not cleave nucleic acids in the telomere sequence. In some cases, the DNA is digested with a restriction enzyme that frequently cleaves within the genomic DNA but not within the telomere sequence. In some cases, the restriction enzyme has 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 repeat sequences and subtelomeric DNA. Subtelomeric DNA is a DNA sequence adjacent to a tandem repeat sequence of the telomere sequence and contains telomeric repeat sequences scattered with variable telomere-like sequences. The digested DNA is separated by electrophoresis and imprinted onto a support (e.g., a membrane). Fragments containing telomere sequences are detected by hybridizing a probe (i.e., a labeled repetitive sequence) to the membrane. After visualization of the telomere-containing fragments, 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). TRF estimation by Southern blotting gives the distribution of telomere length in the cell or tissue, and thus also the median and mean telomere lengths in all cells.

[0154] On the other hand, telomere length can be measured by flow cytometry (Hultdin, M. et al., Nucleic Acid 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 the FISH technique. If the starting material is tissue, a cell suspension is typically prepared by mechanical separation and / or protease treatment. Cells are fixed with a fixative and hybridized with telomere sequence-specific probes (preferably PNA probes) labeled with fluorescent markers. After hybridization, the cells are washed and then analyzed by FACS. The fluorescence signal of cells in Go / G1 is measured after appropriate subtraction of background fluorescence. This technique is suitable for rapid estimation of telomere length in large samples. Similar to TRF, telomere length is the average length of telomeres within the cells.

[0155] In other respects, the median or average length of the telomeres in cells within the 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 product during the PCR reaction causes an increase in fluorescence intensity, which is measured in each cycle of the PCR reaction. This quantifies the DNA concentration. The relative concentration of the DNA present during the exponential phase of the reaction is determined by plotting the fluorescence level versus the number of PCR cycles on a semi-logarithmic scale. A threshold for detecting fluorescence above background is determined. The number of cycles required for fluorescence from the sample to reach the threshold is called the cycle threshold (Ct). Since the amount of DNA theoretically doubles in each cycle of the exponential phase, the relative amount of DNA can be calculated. The baseline is the initial cycle response of the PCR, where the fluorescence signal remains almost unchanged.

[0156] In some aspects, telomere length is determined by telo-FISH. In this method, cells are fixed and hybridized to probes conjugated to fluorescent labels (e.g., Cy-3, fluorescein, rhodamine, etc.). The probes used in this method are oligonucleotides designed for specific hybridization with telomere sequences. Typically, the probes are 8 or more nucleotides long, for example, 12-20 or more nucleotides long. In one aspect, the probes are oligonucleotides containing naturally occurring nucleotides. In another aspect, the probes are peptide nucleic acids that have a higher Tm than similar natural sequences, thus allowing for more stringent hybridization conditions. Cells can be treated with reagents such as colchicine to induce metaphase arrest of the cell cycle, thereby providing metaphase chromosomes for hybridization and analysis. In some embodiments, cellular DNA can also be stained with the fluorescent dye 4',6-diamidinyl-2-phenylindole (DAPI).

[0157] A complete digital image of the metaphase chromosome is obtained, and the fluorescence intensity of the probe hybridizing with telomeres is quantified. This method can measure not only the average or median telomere length of cells, but also the telomere length of individual chromosomes, and avoids problems associated with the presence of subtelomeric DNA (Zjilmans, JM et al., Proceedings of the National Academy of Sciences 94:7423-7428 (1997); Blasco, MA et al., Cell 91:25-34 (1997); incorporated herein by reference). The intensity of the fluorescence signal is correlated with the length of the telomere; a brighter fluorescence signal indicates a longer telomere.

[0158] In some embodiments, the present invention relates to telomerase inhibitors in a method for treating myelofibrosis, the method comprising:

[0159] Identifying patients most likely to benefit from telomerase inhibitor therapy involves performing the following tests on the patients:

[0160] (a) Triple negative status, which is determined based on the absence of any mutations in the JAK2, CALR and MPL genes;

[0161] (b) Polymer risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; or

[0162] (c) Both are present;

[0163] The presence of (a), (b), or (c) indicates that the patient is most likely to benefit from telomerase inhibitor treatment, and an effective amount of telomerase inhibitor is administered to said patient. In some embodiments, the present invention relates to telomerase inhibitors used in the methods defined in any other embodiment.

[0164] Another embodiment of the 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 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 criteria: are triple-negative, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or have 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 patients 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 a telomerase inhibitor for treating myelofibrosis, comprising: (a) screening patients to determine whether such patients are: 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 patients are in a triple-negative state, administering the telomerase inhibitor to the patients.

[0165] Another embodiment of the invention is the use of 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 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 criteria: are triple-negative, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and / or have 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 patients 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 invention is the use of a telomerase inhibitor for treating myelofibrosis, comprising: (a) screening patients to determine whether such patients are: 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 patients are in a triple-negative state, administering the telomerase inhibitor to the patients.

[0166] In some embodiments, a triple-negative status can be determined based on the absence of mutations in each of the following genes: the JAK2 gene having sequence number 2; the CALR gene having sequence number 3; and the MPL gene having sequence number 4. In other embodiments, a 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, a 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, a triple-negative status can be determined based on the absence of mutations in each of the following: JAK2, CALR, and sequence number 4.

[0167] In other embodiments of the invention, polymeric risk (HMR) can be determined based on the presence of mutations 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.

[0168] In other embodiments of the invention, telomerase activity and hTERT expression levels in biological samples obtained from patients can be determined to assess pharmacokinetic effects and / or monitor patients receiving telomerase inhibition therapy. The telomerase activity can be measured using the TRAP (telomere repeat amplification protocol) telomerase activity assay. The hTERT expression level can be determined by measuring the hTERT RNA expression level in the cells within the biological sample using Northern blotting, sequence expression analysis (SAGE), or other methods.

[0169] In some embodiments, the present invention relates to telomerase inhibitors used in the treatment of myelofibrosis as defined in any other embodiment.

[0170] In some embodiments, the present invention relates to the use of telomerase inhibitors in the treatment of myelofibrosis as defined in any other embodiment.

[0171] Other embodiments

[0172] Other embodiments for different purposes are listed in the following terms:

[0173] Clause 1. A method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising:

[0174] (a) Testing the patient for triple-negative status, determined based on the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes; and

[0175] (b) Select the patients who are in a triple-negative state, which is determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes.

[0176] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0177] Clause 2. A method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising:

[0178] (c) Perform the following tests on the patient:

[0179] i. Triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or

[0180] ii. Polymer risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0181] (d) Select the patients who are in the following states:

[0182] i. Triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes, and / or

[0183] ii. Polymer risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0184] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0185] Clause 3. A method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising:

[0186] (e) Obtain DNA samples from the patient;

[0187] (f) Triple-negative status testing of the DNA sample derived from the patient, determined based on the absence of mutations in each of JAK2, CALR, and MPL; and

[0188] (g) Select patients who meet the following criteria and are in a triple-negative state, which is determined based on the absence of mutations in each of the JAK2, CALR and MPL genes;

[0189] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0190] Clause 4. A method for identifying patients most likely to benefit from telomerase inhibitor treatment, comprising:

[0191] (h) Obtain DNA samples from patients;

[0192] (i) Perform the following tests on the DNA sample derived from the patient:

[0193] i. Triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and / or

[0194] ii. Polymer risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2; and

[0195] (j) Select the patients who meet the following criteria:

[0196] i. Triple-negative status, determined based on the absence of mutations in each of the JAK2, CALR, and MPL genes; and / or

[0197] ii. Polymer risk (HMR), determined based on the presence of mutations in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1 / 2.

[0198] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0199] Clause 5. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein the patients are determined to be triple-negative, 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.

[0200] Clause 6. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein said patients are identified as having high molecular risk (HMR).

[0201] The mutations in genes with HMRs include at least one group selected from the following: extra comb-like protein 1 (ASXL1), Zeste gene enhancer homolog 2 (EZH2), serine and arginine enriched splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0202] Clause 7. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein the mean relative telomere length of cells present in a biological sample of said patient is determined to be at or below the 50th percentile of the range of relative telomere lengths (determined according to one or more known criteria).

[0203] Clause 8. Use of telomerase inhibitors in the manufacture of medicaments for the treatment of patients with myelofibrosis, wherein the patients are determined to be in a triple-negative state, wherein the triple-negative state comprises the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes.

[0204] Clause 9. Use of telomerase inhibitors in the manufacture of medicaments for the treatment of patients with myelofibrosis, wherein said patients are identified as having a high molecular risk (HMR), wherein the HMR comprises a mutation in at least one gene selected from the group consisting of: extra comb-like protein 1 (ASXL1), Zeste gene enhancer homolog 2 (EZH2), serine and arginine enriched splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0205] Clause 10. Use of telomerase inhibitors in the manufacture of medicaments for the treatment of patients with myelofibrosis, wherein the mean relative telomere length of cells present in a biological sample of said patient is determined to be at or below the 50th percentile of the range of relative telomere lengths (determined according to one or more known criteria).

[0206] The following examples are for illustrative purposes only and are not intended to be limiting.

[0207] Example

[0208] Example 1 : Imesta sodium is indicated for patients with relapsed disease or those resistant to Janus kinase (JAK) inhibitor therapy. Effective treatment for patients with intermediate-2 (Int-2) or high-risk myelofibrosis (MF)

[0209] introduce

[0210] Imesta, a 13-meric oligonucleotide that specifically targets the RNA template of human telomerase, is a potent competitive inhibitor of telomerase activity (Asai et al., Cancer Research, 2003; Herbert, Oncogenes, 2005). Clinical activity and acceptable safety profiles 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 inhibitors (JAKi) (Tefferi, New England Journal of Medicine, 2015). This example provides results from a phase 2 clinical study of ememsta sodium in patients with myelofibrosis (MF) at two dose levels.

[0211] method

[0212] A randomized, multicenter phase 2 study was conducted in adults meeting the following criteria using two doses of emexert sodium (9.4 mg / kg or 4.7 mg / kg, intravenously, every 3 weeks): a Dynamic International Prognostic Score (DIPSS) int-2; or high-risk myelofibrosis (MF) previously treated with JAKi but now relapsed / resistant (i.e., no reduction in splenomegaly within 12 weeks of initiation of JAK inhibitor (“JAKi”) treatment; or worsening of splenomegaly at any time after initiation of said treatment). A diagnosis of primary, essential thrombocythemia post-myelofibrosis, or polycythemia vera post-myelofibrosis was required; other eligibility criteria included measurable splenomegaly (measured by magnetic resonance imaging [MRI]), active MF-related systemic symptoms, and a platelet count ≥75 x 10⁻⁶. 9 / L. The primary endpoints were: spleen response rate (spleen volume reduction [SVR] ≥35% as measured by MRI at week 24); and symptom relief rate (total symptom score [TSS] ≥50% as measured by the Myelofibrosis Symptom Assessment Scale (MFSAF) v2 at week 24). Key secondary endpoints included safety, overall survival (OS), treatment response, molecular response, and pharmacokinetic and pharmacodynamic relationships.

[0213] result

[0214] 107 patients were recruited from 55 institutions (48 received a dose of 4.7 mg / kg; 59 received a dose of 9.4 mg / kg). Baseline characteristics are shown in Table 1 below. Furthermore, the median duration of JAKi treatment was 23 (0.9–89.7) months, and the median platelet count was 147 x 10⁻⁶. 9 / L. 24.8% of patients had triple-negative results (TN; i.e., no mutations in JAK2, MPL, or CALR), while 67.6% of patients were considered to have high molecular risk (HMR; i.e., ≥1 mutation in ASXL1, EZH2, SRSF2, or IDH1 / 2).

[0215]

[0216] At the end of the initial clinical trial, 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). The IRC confirmed, based on MRI, that splenic responses occurred in 6 patients (10.2%) in the 9.4 mg / kg treatment group.

[0217] At the end of the clinical trial, patients were followed up for a median duration 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 in the 9.4 mg / kg group (7.7 months) was longer than that in the 4.7 mg / kg group. Based on MRI, splenic responses were observed in 6 patients (10.2%) in the 9.4 mg / kg group, while no responses were observed in the 4.7 mg / kg group (see [link to MRI]). Figure 1 In the 9.4 mg / kg treatment group and the 4.7 mg / kg treatment group, 19 patients (32%) and 3 patients (6%), respectively, experienced symptomatic responses (TSS reduction ≥50%) (see...). Figure 2 ).

[0218] At the end of the first clinical trial, the median overall survival (OS) was not reached in the 9.4 mg / kg treatment group, while the median OS in the 4.7 mg / kg treatment group was 19.9 months. 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. Sensitivity analyses were performed on patients undergoing dose escalation to inform subsequent JAKi treatment or stem cell transplantation for similar outcomes. An association between TN (tumor nephropathy) and OS was observed in the 9.4 mg / kg treatment group (the median OS was not reached in TN patients, compared to 23.6 months in non-TN patients). Patients carrying one HMR mutation (ASXL1, EZH2, SRSF2, or IDH1 / 2) showed higher splenic response rates.

[0219] 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 group (13% and 29%, respectively); most cytopenia resolved within 4 weeks. Grade 3 / 4 LFT elevation was observed in 7 patients at the time of the study. No emexeter-related hepatotoxicity confirmed by the independent liver review committee was observed.

[0220] At the end of the second clinical trial, patients were followed up for a median follow-up of 27.4 (0.2–33.0) months, including a median treatment duration of 26.9 (0.1–118.1) weeks. The median treatment duration in the 9.4 mg / kg group (33.3 weeks) was longer than that in the 4.7 mg / kg group (23.9 weeks). The early termination of the study in the 4.7 mg / kg group affected the treatment duration. In the 9.4 mg / kg group, the median overall survival (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 trial.

[0221] Triple Negative and OS

[0222] Subjects were grouped according to their mutation status in the JAK2 / MPL / CALR genes—triple-negative (TN, no mutation in any of the JAK2 / MPL / CALR genes) and non-TN (any mutation in any of the JAK2 / MPL / CALR genes). In the 9.4 mg / kg treatment group, the median overall survival (OS) was not estimable (NE) for TN subjects (95% confidence interval) (23.2, NE), and for non-TN subjects (95% confidence interval) (20.7, NE) it was 23.6 months. In the 4.7 mg / kg treatment group, the median OS (95% confidence interval) for TN subjects was 22.3 (17, NE) and for non-TN subjects was 20.3 (18.3, NE), respectively. In the 9.4 mg / kg treatment group, the triple-negative (TN) group had a lower mortality rate compared to the non-TN group (see Table 2). Figure 3 and 4 ).

[0223]

[0224] At the end of the second clinical trial, in the 9.4 mg / kg treatment group, the mortality rate was lower in the triple-negative (TN) group compared to the non-TN group (see Table 3). Figure 6 and 7 ).

[0225] Table 3: Overall survival grouped according to the mutation status of JAK2 / MPL / CALR genes

[0226]

[0227] *HR = Risk Ratio

[0228] Relationship between negative result on Wednesday of week 24 and response

[0229] 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).

[0230] Table 4: Baseline JAK2 / CALR / MPL mutation status categorized by clinical response

[0231]

[0232]

[0233] Relationship between molecular risk and response in week 24

[0234] 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).

[0235] Table 5: Relationship between molecular risk and response at week 24

[0236]

[0237]

[0238] In subjects receiving a dose of 9.4 mg / kg, the following factors were observed to be associated with clinical response or overall survival (OS):

[0239] Triple negative (TN): Enhanced response (SVR or TSS) was observed in TN subjects. The median overall survival (OS) in the TN group was not estimable, while the median OS in the non-TN group was 23.6 months; and

[0240] Molecular risks: Enhanced response (SVR or TSS) was observed in HMR subjects with only one mutation, and corresponding responses were also observed in HMR patients with more than one mutation who received 9.4 mg / kg imelt.

[0241] Example 2

[0242] Baseline telomere length (TL) and overall survival (OS)

[0243] Subjects were grouped according to the median baseline TL. In the 9.4 mg / kg treatment group, at the end of the first clinical study, the median OS was not estimable (95% confidence interval) for subjects with shorter baseline TLs (<= median) (23.2, NE); while for subjects with longer baseline TLs (> 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 shorter baseline TLs and longer TLs were 20.3 (17.2, NE) months and 22.3 (16.6, NE) months, respectively (Table 6).

[0244] In the 9.4 mg / kg treatment group, a better trend toward overall survival (OS) was observed in subjects with shorter baseline total retention (TL) (i.e., baseline TL less than or equal to the median TL).

[0245] Table 6: Overall survival based on baseline telomere length (TL) grouped by median

[0246]

[0247] Relationship between baseline TL and response at week 24

[0248] Baseline telomere length (TL): At week 24, subjects with shorter baseline TLs (<= median) showed enhanced SVR or TSS responses. Splenic responses occurred in 17.3% (5 / 29) of subjects with shorter baseline TLs and 4.2% (1 / 24) of subjects with longer baseline TLs. TSS responses occurred in 34.5% (10 / 29) of subjects with shorter baseline TLs and 25% (6 / 24) of subjects with longer baseline TLs.

[0249] At week 24, in the 9.4 mg / kg treatment group, subjects with shorter baseline time intervals (TLs) showed higher response rates (SVR or TSS) compared to subjects with longer TLs (Table 7).

[0250] Table 7: Baseline summary of telomere length (TL) categorized by clinical response

[0251]

[0252] Example 3

[0253] Dose-dependent pharmacodynamic (PD) effects

[0254] Telomerase activity and hTERT were analyzed to assess the pharmacokinetic effects of imeresta. In subjects with available baseline and post-treatment data, telomerase activity decreased by ≥50% from baseline in 23 subjects (51.1%) and 10 subjects (29.4%) in the 9.4 mg / kg and 4.7 mg / kg treatment groups, respectively, indicating that PD effects were associated with antitumor activity in preclinical xenograft models. Additionally, hTERT RNA levels decreased by ≥50% from baseline in 35 subjects (61.4%) and 20 subjects (47.7%) in the 9.4 mg / kg and 4.7 mg / kg treatment groups, respectively (Table 8). This confirmed dose-dependent PD effects, indicating target involvement.

[0255] Table 8: Reduction of TA (30%), 50%) or hTERT (50%) relative to baseline achieved by subjects at any time point.

[0256]

[0257] Example 4

[0258] Association between PD effects and responses at week 24

[0259] Compared with non-splenic responders (55.6%), a higher proportion of subjects in splenic responders (83.3%) had a reduction in hTERT RNA expression levels of at least ≥50%; compared with non-TSS responders, a higher proportion of subjects in TSS-responding subjects had a reduction in hTERT RNA expression levels of at least ≥50% (Table 9). hTERT RNA expression levels were measured using whole blood samples collected from patients before and after treatment.

[0260] Table 9: Association between the decrease (50%) of hTERT relative to baseline at week 24 and SVR or TSS response.

[0261]

[0262] Compared with subjects who did not have a spleen response or a TSS response, a higher proportion of subjects who did have a spleen response or a TSS response had a reduction in telomerase activity of at least 30% or 50% (Table 10).

[0263] Table 10: Association between decreases in telomerase activity relative to baseline (30%, 50%) at week 24 and SVR or TSS response.

[0264]

[0265]

[0266] The 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 scope of this invention, certain non-limiting aspects of the invention are provided below. Upon reading this invention, the following will be apparent to those skilled in the art: each individually numbered content may be used alone or in combination with any preceding or following individually numbered content. This is intended to support all such combinations of the content and is not limited to combinations of the content explicitly provided below:

[0267] 1. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein the patients are determined to be triple-negative.

[0268] The triple-negative state refers to the absence of mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes.

[0269] 2. The use according to 1, wherein the myelofibrosis is primary myelofibrosis.

[0270] 3. According to the use described in 2, the myelofibrosis described therein is post-PV MF.

[0271] 4. According to the use described in 2, the myelofibrosis is post-ET MF (post-ET myelofibrosis).

[0272] 5. The use according to any one of 1-4, wherein the patient has not previously received JAK inhibitor therapy.

[0273] 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.

[0274] 7. The use according to any one of 1-4, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed.

[0275] 8. The use according to any one of 1-4, wherein the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0276] 9. The use according to any one of 1-8, wherein the telomerase inhibitor is imestat.

[0277] 10. The use according to 9, wherein the imesta is imesta sodium.

[0278] 11. The use according to 10, wherein the telomerase inhibitor is imestat, and is administered in 1, 2, 3, 4, 5, 6, 7, 8 or more dosing cycles, each cycle comprising:

[0279] The intravenous administration of approximately 7-10 mg / kg imestat is given once every three weeks.

[0280] The intravenous administration of approximately 7-10 mg / kg imestat is once a week for three weeks.

[0281] Administer intravenously at approximately 2.5-10 mg / kg of imestat every three weeks; or

[0282] The intravenous administration is approximately 0.5-9.4 mg / kg of imestat, once every three weeks.

[0283] 12. As described in 11, each dosing cycle comprises an intravenous administration of approximately 7-10 mg / kg imestat every three weeks.

[0284] 13. As described in 12, wherein each dosing cycle comprises an intravenous administration of approximately 9.4 mg / kg imestat once every three weeks.

[0285] 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 present in target cells within the biological sample of the patient.

[0286] 15. The use according to any one of 1-14 further comprises selecting patients whose mean relative telomere length in target cells present in the corresponding biological sample is determined to be at or below the 50th percentile of the relative telomere length range (determined according to one or more known criteria).

[0287] 16. The use according to any one of 1-15 further comprises screening a patient to determine whether the patient has a high molecular risk (HMR), wherein having an HMR comprises a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2 and IDH1 / 2.

[0288] 17. The use according to any one of 1-16 further includes assessing the level of hTERT expression in biological samples obtained from the patient after administration of the telomerase inhibitor.

[0289] 18. The use as described in 17, wherein the hTERT expression level is reduced by 50% or more relative to the baseline hTERT expression level prior to administration of the telomerase inhibitor.

[0290] 19. The use according to any one of 17-18 further comprises changing the dose, frequency of administration, and treatment procedure of the telomerase inhibitor applied to the subject.

[0291] 20. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein said patients are identified as having high molecular risk (HMR).

[0292] The mutations in genes with HMRs include at least one group selected from the following: extra comb-like protein 1 (ASXL1), Zeste gene enhancer homolog 2 (EZH2), serine and arginine enriched splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1 / 2 (IDH1 / 2).

[0293] 21. The use according to 20, wherein the myelofibrosis is primary myelofibrosis.

[0294] 22. According to the use described in 21, wherein the myelofibrosis is post-PV MF.

[0295] 23. According to the use described in 21, the myelofibrosis is post-ET myelofibrosis (post-ET MF).

[0296] 24. The use according to any one of 20-23, wherein the patient has not previously received JAK inhibitor therapy.

[0297] 25. The use according to any one of 20-23, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy.

[0298] 26. The use according to any one of 20-23, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed.

[0299] 27. The use according to any one of 20-23, wherein the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0300] 28. The use according to any one of 20-27, wherein the telomerase inhibitor is imestat.

[0301] 29. The use according to 28, wherein the imesta is imesta sodium.

[0302] 30. The use according to 28, wherein the telomerase inhibitor is imelistar, and is administered in 1, 2, 3, 4, 5, 6, 7, 8 or more dosing cycles, each cycle comprising:

[0303] The intravenous administration of approximately 7-10 mg / kg imestat is given once every three weeks.

[0304] The intravenous administration of approximately 7-10 mg / kg imestat is once a week for three weeks.

[0305] Administer intravenously at approximately 2.5-10 mg / kg of imestat every three weeks; or

[0306] The intravenous administration is approximately 0.5-9.4 mg / kg of imestat, once every three weeks.

[0307] 31. As described in 30, each dosing cycle comprises an intravenous administration of approximately 7-10 mg / kg imestat every three weeks.

[0308] 32. As described in 31, wherein each dosing cycle comprises an intravenous administration of approximately 9.4 mg / kg imestat once every three weeks.

[0309] 33. The use according to any one of 20-32 further comprises determining the average relative telomere length by analyzing the relative length of telomere nucleic acids in target cells present within the biological sample of the patient.

[0310] 34. The use according to any one of 20-33 further comprises selecting patients whose mean relative telomere length in target cells present in the corresponding biological sample is determined to be at or below the 50th percentile of the relative telomere length range (determined according to one or more known criteria).

[0311] 35. The use according to any one of 20-34 further comprises screening a 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.

[0312] 36. The use according to any one of 20-35 further includes assessing the level of hTERT expression in biological samples obtained from the patient after administration of the telomerase inhibitor.

[0313] 37. The use as described in 36, wherein the hTERT expression level is reduced by 50% or more relative to the baseline hTERT expression level prior to administration of the telomerase inhibitor.

[0314] 38. The use according to any one of 36-37 further comprises altering the dose, frequency of administration, and treatment process of the telomerase inhibitor applied to the subject.

[0315] 39. Use of telomerase inhibitors in the treatment of patients with myelofibrosis, wherein the mean relative telomere length of cells present in a biological sample of said patient is determined to be the 50th percentile or lower of the range of relative telomere lengths (determined according to one or more known criteria).

[0316] 40. The use according to 39, wherein the myelofibrosis is primary myelofibrosis.

[0317] 41. According to the use described in 40, wherein the myelofibrosis is post-PV MF.

[0318] 42. According to the use described in 40, the myelofibrosis is post-ET myelofibrosis (post-ET MF).

[0319] 43. The use according to any one of 39-42, wherein the patient has not previously received JAK inhibitor therapy.

[0320] 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.

[0321] 45. The use according to any one of 39-42, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed.

[0322] 46. ​​The use according to any one of 39-42, wherein the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0323] 47. The use according to any one of 39-46, wherein the telomerase inhibitor is imestat.

[0324] 48. The use according to 47, wherein the imesta is imesta sodium.

[0325] 49. The use according to 47, wherein the telomerase inhibitor is imelistar, and is administered in 1, 2, 3, 4, 5, 6, 7, 8 or more dosing cycles, each cycle comprising:

[0326] The intravenous administration of approximately 7-10 mg / kg imestat is given once every three weeks.

[0327] The intravenous administration of approximately 7-10 mg / kg imestat is once a week for three weeks.

[0328] Administer intravenously at approximately 2.5-10 mg / kg of imestat every three weeks; or

[0329] The intravenous administration is approximately 0.5-9.4 mg / kg of imestat, once every three weeks.

[0330] 50. As described in 49, each dosing cycle comprises an intravenous administration of approximately 7-10 mg / kg imestat every three weeks.

[0331] 51. As described in 50, each dosing cycle comprises an intravenous administration of approximately 9.4 mg / kg imestat every three weeks.

[0332] 52. The use according to any one of 39-51 further comprises determining the average relative telomere length by analyzing the relative length of telomere nucleic acids present in the cells within the biological sample of the patient.

[0333] 53. The use according to any one of 39-52 further includes assessing the level of hTERT expression in biological samples obtained from the patient after administration of the telomerase inhibitor.

[0334] 54. According to the use described in 53, wherein the hTERT expression level is reduced by 50% or more relative to the baseline hTERT expression level before the administration of the telomerase inhibitor.

[0335] 55. The use according to any one of 53-54 further comprises altering the dose, frequency of administration, and treatment procedure of the telomerase inhibitor applied to the subject.

[0336] 56. A method for selecting patients most likely to benefit from telomerase inhibitor therapy, comprising:

[0337] The patient was tested for triple-negative status, wherein the triple-negative status included the absence of mutations in each of the JAK2, CALR, and MPL genes; and

[0338] Select the patients who are in a triple-negative state.

[0339] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0340] 57. The method according to 56, wherein the patient suffers from myelofibrosis.

[0341] 58. The method according to 57, wherein the myelofibrosis is primary myelofibrosis.

[0342] 59. The method according to 57, wherein the myelofibrosis is post-PV MF.

[0343] 60. The method according to 57, wherein the myelofibrosis is post-ET MF.

[0344] 61. The method according to any one of 56 to 60, wherein the patient has not previously received JAK inhibitor therapy.

[0345] 62. The method according to any one of 56 to 60, wherein the patient:

[0346] He had previously received JAK inhibitor therapy;

[0347] Previously treated with failed JAK inhibitor therapy; or

[0348] The patient had previously received JAK inhibitor therapy and discontinued it due to treatment-related toxicity or intolerance.

[0349] 63. The 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.

[0350] 64. The method according to any one of 56 to 60, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed.

[0351] 65. The method according to any one of 56 to 60, wherein the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0352] 66. The method according to any one of 56 to 65 further comprises administering a telomerase inhibitor to the patient.

[0353] 67. The method according to 66, wherein the telomerase inhibitor is imestat.

[0354] 68. The method according to 67, wherein the imesta is imesta sodium.

[0355] 69. The method according to any one of 56-68, further comprising obtaining a sample (containing DNA) from the patient.

[0356] 70. The method of claim 69, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof.

[0357] 71. The method according to 70, wherein the step of obtaining a sample from the patient comprises:

[0358] Obtain bone marrow samples, peripheral blood samples, or combinations thereof; and

[0359] DNA was isolated from the bone marrow sample, peripheral blood sample, or a combination thereof.

[0360] 72. The method of 70, wherein the step of obtaining a sample from the patient comprises:

[0361] Obtain bone marrow samples from the patients;

[0362] Cells were isolated from the bone marrow sample; and

[0363] DNA was extracted from the isolated cells.

[0364] 73. The method of 70, wherein the step of obtaining a sample from the patient comprises:

[0365] Peripheral blood samples were obtained from the patients.

[0366] Cells were isolated from the peripheral blood sample; and

[0367] DNA was extracted from the isolated cells.

[0368] 74. A method for selecting patients most likely to benefit from telomerase inhibitor therapy, comprising:

[0369] The patient is tested to determine whether the patient has HMR, wherein having HMR involves the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2, and IDH1 / 2; and

[0370] Select the patients with HMR.

[0371] The selected patients are the most likely to benefit from telomerase inhibitor therapy.

[0372] 75. The method according to 74, wherein the patient suffers from myelofibrosis.

[0373] 76. The method according to 75, wherein the myelofibrosis is primary myelofibrosis.

[0374] 77. The method according to 75, wherein the myelofibrosis is post-PV myelofibrosis (post-PV MF).

[0375] 78. The method according to 75, wherein the myelofibrosis is post-ET MF.

[0376] 79. The method according to any one of 74 to 78, wherein the patient has not previously received JAK inhibitor therapy.

[0377] 80. The method according to any one of 74 to 78, wherein the patient:

[0378] He had previously received JAK inhibitor therapy;

[0379] Previously treated with failed JAK inhibitor therapy; or

[0380] The patient had previously received JAK inhibitor therapy and discontinued it due to treatment-related toxicity or intolerance.

[0381] 81. The method according to any one of 74 to 78, wherein the patient has received JAK inhibitor therapy and the patient is resistant to JAK inhibitor therapy.

[0382] 82. The method according to any one of 74 to 78, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed.

[0383] 83. The method according to any one of 74 to 78, wherein the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0384] 84. The method according to any one of 74 to 83 further comprises administering a telomerase inhibitor to the patient.

[0385] 85. The method according to 84, wherein the telomerase inhibitor is imestat.

[0386] 86. The method according to 85, wherein the imesta is imesta sodium.

[0387] 87. The method according to any one of 74-86 further comprises obtaining a sample (containing DNA) from the patient.

[0388] 88. The method according to 87, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof.

[0389] 89. The method according to 88, wherein the step of obtaining a sample from the patient comprises:

[0390] Obtain bone marrow samples, peripheral blood samples, or combinations thereof; and

[0391] DNA was isolated from the bone marrow sample, peripheral blood sample, or a combination thereof.

[0392] 90. The method according to 88, wherein the step of obtaining a sample from the patient comprises:

[0393] Obtain bone marrow samples from the patients;

[0394] Cells were isolated from the bone marrow sample; and

[0395] DNA was extracted from the isolated cells.

[0396] 91. The method according to 88, wherein the step of obtaining a sample from the patient comprises:

[0397] Peripheral blood samples were obtained from the patients.

[0398] Cells were isolated from the peripheral blood sample; and

[0399] DNA was extracted from the isolated cells.

[0400] 92. A method for selecting patients most likely to benefit from telomerase inhibitor treatment, the method comprising:

[0401] The average relative telomere length of the patient was tested, which was performed by analyzing the relative length of telomere nucleic acids present in target cells within the patient's biological sample; and

[0402] Patients whose mean relative telomere length in target cells present in the corresponding biological sample is determined to be at or below the 50th percentile of the relative telomere length range (determined according to one or more known criteria) are selected, wherein the selected patients are most likely to benefit from telomerase inhibitor treatment.

[0403] 93. The method according to 92, wherein the patient suffers from myelofibrosis.

[0404] 94. The method according to 93, wherein the myelofibrosis is primary myelofibrosis.

[0405] 95. The method according to 93, wherein the myelofibrosis is post-PV MF.

[0406] 96. The method according to 93, wherein the myelofibrosis is post-ET MF.

[0407] 97. The method according to any one of 92 to 96, wherein the patient has not previously received JAK inhibitor therapy.

[0408] 98. The method according to any one of 92 to 96, wherein the patient:

[0409] He had previously received JAK inhibitor therapy;

[0410] Previously treated with failed JAK inhibitor therapy; or

[0411] The patient had previously received JAK inhibitor therapy and discontinued it due to treatment-related toxicity or intolerance.

[0412] 99. The 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.

[0413] 100. The method according to any one of 92 to 96, wherein the patient has received JAK inhibitor therapy and the corresponding disease has relapsed.

[0414] 101. The method according to any one of 92 to 96, wherein the patient has received JAK inhibitor therapy and has now discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

[0415] 102. The method according to any one of 92 to 101 further comprises administering a telomerase inhibitor to the patient.

[0416] 103. The method according to 102, wherein the telomerase inhibitor is imestat.

[0417] 104. The method according to 103, wherein the imesta is imesta sodium.

[0418] 105. The method according to any one of 92-104, further comprising obtaining a sample (containing DNA) from the patient.

[0419] 106. The method of 105, wherein the sample comprises bone marrow, peripheral blood, or a combination thereof.

[0420] 107. The method of 106, wherein the step of obtaining a sample from the patient comprises:

[0421] Obtain bone marrow samples, peripheral blood samples, or combinations thereof; and

[0422] DNA was isolated from the bone marrow sample, peripheral blood sample, or a combination thereof.

[0423] 108. The method of 106, wherein the step of obtaining a sample from the patient comprises:

[0424] Obtain bone marrow samples from the patients;

[0425] Cells were isolated from the bone marrow sample; and

[0426] DNA was extracted from the isolated cells.

[0427] 109. The method of 106, wherein the step of obtaining a sample from the patient comprises:

[0428] Peripheral blood samples were obtained from the patients.

[0429] Cells were isolated from the peripheral blood sample; and

[0430] DNA was extracted from the isolated cells.

[0431] 110. A method for monitoring the effect of treatment in subjects with myelofibrosis (MF), the method comprising:

[0432] After administration of a telomerase inhibitor, hTERT expression levels were measured in biological samples obtained from the patients; and

[0433] The hTERT expression level in the biological sample was compared with the baseline hTERT expression level before the administration of the telomerase inhibitor.

[0434] A 50% or greater reduction in hTERT expression levels in the biological sample may identify subjects with an increased likelihood of benefiting from treatment with the telomerase inhibitor.

[0435] 111. The method according to 110, wherein the measured or evaluated hTERT expression level is the hTERT RNA expression level.

[0436] 112. A method for identifying patients with myelofibrosis (MF) suitable for treatment with telomerase inhibitors, the method comprising:

[0437] After administration of a telomerase inhibitor, hTERT expression levels were measured in biological samples obtained from the patients; and

[0438] The hTERT expression level in the biological sample was compared with the baseline hTERT expression level before the administration of the telomerase inhibitor.

[0439] The reduced hTERT expression level in the biological sample may identify patients who are more likely to benefit from treatment with the telomerase inhibitor.

[0440] 113. The method according to 112, wherein the hTERT expression level is reduced by 50% or more.

[0441] Although the specific embodiments have been described in detail with illustrations and examples for the purpose of clarity, it will be apparent, given the edifying significance of the invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0442] Therefore, the foregoing only illustrates the principles of the invention. Various structures can be designed, although not explicitly stated or shown herein, but these designs reflect the principles of the invention and do not depart from its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to help the reader understand the principles of the invention and the inventors' ideas for further expanding the field, and should be interpreted as not being limited by these specifically listed examples and conditions. Moreover, all statements herein referencing the principles, aspects, and embodiments of the invention and their specific examples are intended to cover their structural and functional equivalents. Furthermore, the equivalents are intended to include currently known equivalents and those to be developed in the future, i.e., any functionally identical elements developed regardless of their structure. Therefore, the scope of the invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. sequence list <110> American Jerome Biopharmaceuticals J.C. Busolari Huang Fei <120> Methods for identifying patients who may benefit from telomerase inhibitor therapy <130> GERN-192 <160> 9 <170> PatentIn Version 3.5 <210> 1 <211> 13 <212> DNA <213> Synthetic oligonucleotides <400> 1 tagggttaga caa 13 <210> 2 <211> 142939 <212> DNA <213> JAK2 oligonucleotides <400> 2 142939 <210> 3 <211> 5883 <212> DNA <213> CALR oligonucleotides <400> 3 5883 <210> 4 <211> 14996 <212> DNA <213> MPL oligonucleotides <400> 4 14966 <210> 5 <211> 80968 <212> DNA <213> ASXL1 oligonucleotide <400> 5 80968 Serial Number: 6 EZH2 Serial Number: 7 SRSF2 Serial Number: 8 IDH1 Serial Number: 9 IDH2

Claims

1. Use of a telomerase inhibitor in the preparation of a medicament for treating a patient with myelofibrosis, wherein the patient is determined to be in a triple negative state, wherein the triple negative state refers to JAK2 , CALR and MPL There are no mutations in any of the genes, said patient determined to be of said triple negative status has an increased likelihood of benefiting from treatment with said telomerase inhibitor; The telomerase inhibitor is imetelstat or imetelstat sodium.

2. 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).

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

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

5. The use according to claim 1, wherein The patient is administered the telomerase inhibitor when being determined to be triple negative.

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