Methods of treating myelodysplastic syndromes and monitoring treatment

Abstract

Provided is a method for monitoring the effectiveness of treatment in a subject with myelodysplastic syndrome (MDS). Also provided is a method for identifying a subject with MDS for treatment with a telomerase inhibitor, and a method for treating MDS. The method includes administering a telomerase inhibitor to a subject, and evaluating the variant allele frequency (VAF) of one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1 in a biological sample obtained from the subject after administration of the telomerase inhibitor. In some cases, a reduction in VAF of 25% or more identifies a subject who is likely to benefit from treatment with a telomerase inhibitor. In one example, the telomerase inhibitor is imetelstat or imetelstat sodium.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 436,831 filed January 3, 2023, U.S. Provisional Application No. 63 / 444,789 filed February 10, 2023, U.S. Provisional Application No. 63 / 448,638 filed February 27, 2023, U.S. Provisional Application No. 63 / 503,896 filed May 23, 2023, U.S. Provisional Application No. 63 / 505,918 filed June 2, 2023, U.S. Provisional Application No. 63 / 520,538 filed August 18, 2023, and U.S. Provisional Application No. 63 / 606,256 filed December 5, 2023, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Introduction Myelodysplastic syndromes (MDS) are a group of conditions, including cancers of the blood and bone marrow. These include diseases such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with polycytic dysplasia, refractory cytopenia with monocytic dysplasia, and chronic myelomonocytic leukemia. MDS is a group of hematological medical conditions involving inefficient production of myeloid classes of blood cells. In MDS, immature blood stem cells (blasts) do not develop into healthy red blood cells, white blood cells, or platelets. The blasts die in the bone marrow or migrate to the blood and then die soon after, leaving less room in the bone marrow for healthy white blood cells, red blood cells, and / or platelets to form. [Background technology]

[0003] MDS primarily affects elderly individuals and is characterized by anemia and other cytopenias, as well as a high risk of developing leukemia (Cheson et al., Blood 2006;108:419-425). In clinical practice, MDS is suspected when otherwise unexplained anemia is associated with other cytopenias, increased mean cell volume, or increased red cell distribution width. Diagnosis involves bone marrow examination and cytogenetic testing. The bone marrow is typically hyperproliferative. Diagnosis is based on the demonstration of erythroid, granulocytic, or megakaryocytic dysplasia in 10% or more of the signaling cells (Vardiman, et al., Blood 2009;114(5):937-951). MDS can progress over time. For example, patients with MDS often develop severe anemia, requiring frequent blood transfusions. The risk of bleeding and infection also occurs due to low or dysfunctional platelets and neutrophils, respectively. In some cases, the disease worsens, and patients develop cytopenia (decreased blood cell counts) caused by progressive bone marrow failure. In other cases, the disease transforms into acute myeloid leukemia (AML). Transformation to AML is said to have occurred when the overall percentage of bone marrow blasts exceeds a certain cutoff (20% for the World Health Organization (WHO) and 30% for the French-American-British (FAB) subtype). Patients with lower-risk MDS who are relapsed or refractory to erythropoiesis-stimulating agents (ESAs) have limited treatment options.

[0004] The standard prognostic tool for assessing MDS is the International Prognostic Scoring System (IPSS), which classifies patients into low-risk, intermediate-1-risk, intermediate-2-risk, and high-risk categories based on several prognostic variables, including bone marrow blasts, cytogenetics, and the presence of cytopenias. The median survival times for these four groups have been estimated to be 5.7, 3.5, 1.2, and 0.4 years, respectively. The median time to AML development in 25% of patients in these groups was 9.4, 3.3, 1.1, and 0.2 years, respectively (Greenberg et al., Blood 1997;89(6):2079-2088). Patients with low-risk and intermediate-1-risk MDS are sometimes referred to as having "lower-risk" disease, while patients with intermediate-2-risk and high-risk MDS are sometimes referred to as having "higher-risk" disease.

[0005] In Western countries, the incidence of MDS in patients aged 70 years or older is conservatively estimated at approximately 30-40 cases per 100,000 people per year. Due to the aging of the population, the number of MDS cases is expected to increase. Although the rate of leukemia in lower-risk patients is decreasing, most patients are affected by anemia and anemia-related symptoms, which significantly impact patient-reported outcomes (Almeida et al., Leukemia Res. 2017;52:50-57). Many anemic patients with MDS ultimately develop dependence on red blood cell ("RBC") transfusions ("transfusion dependency"). Evidence suggests that iron overload resulting from chronic RBC transfusions may contribute to the overall mortality of this disease (Malcovati et al., J Clin Oncol 2005;23:7594-7603; Malcovati et al., Haematologica 2006;91:1588-1590; Steensma DP., Mayo Clinic Proc. 2015;90(7):969-983). A retrospective analysis of data from 426 patients diagnosed with MDS according to WHO criteria in Italy between 1992 and 2004 showed that requiring two units of blood transfusion per month reduced life expectancy in MDS patients by approximately 50% (Malcovati et al., Haematologica 2006).

[0006] Treatment strategies for MDS are primarily based on the IPSS score. For patients classified as IPSS intermediate-2 or high-risk (higher-risk MDS) and with a median survival of only approximately 12 months if untreated, the goals of treatment are to modify the disease course, avoid progression to AML, and prolong survival. For patients classified as IPSS low-risk or intermediate-1 (lower-risk MDS), survival is longer, but many patients die of causes other than MDS. Treatment for these patients is primarily aimed at improving cytopenias and transfusion outcomes and improving quality of life (Ades et al., Lancet 2014;383(9936):2239-2252).

[0007] For patients with low-risk non-del(5q) MDS, first-line treatment of anemia often involves the use of ESAs or other hematopoietic growth factors. High-dose ESAs (e.g., epoetin alfa), with or without granulocyte colony-stimulating factor, have resulted in erythroid response rates ranging from 30% to 50% and lasting a median of 2 years (ibid.). Key favorable prognostic factors for response to ESAs are low or absent RBC transfusion requirements (<2 packed red blood cell units / month) and low serum erythropoietin levels (<500 units / L) (Hellstrom-Lindberg et al., Br J Haematol. 2003;120(6):1037-1046). Studies have shown that ESAs do not affect the risk of progression to higher-risk MDS and AML and strongly suggest that they may even improve survival in low-risk MDS compared with RBC transfusion alone (Garcia-Manero et al., J Clin Oncol. 2011;29(5):516-523). In the absence of concurrent progression to higher-risk MDS or AML, patients who were initially refractory to ESAs or who relapsed within 6 months of achieving a response had a relatively high risk of AML transformation (23.1%) and shorter survival (median 3 years), whereas patients who responded to treatment and relapsed after 6 months had a more favorable outcome after failure, with a 9% 7-year AML risk and a median overall survival of 4.5 years (Kelaidi et al., Leukemia 2013;27(6):1283-1290).

[0008] In the United States, there are no approved therapies for patients with low-risk non-del(5q) MDS who do not respond to ESAs, and treatment options after ESA failure are limited. Most patients with lower-risk MDS ultimately require long-term RBC transfusions, which are often accompanied by iron overload (Ades et al., Lancet 2014; Fenaux et al., Blood 2013;121:4280-4286; Steensma et al., Mayo Clinic Proc. 2015). Life expectancy in MDS patients has been shown to be inversely proportional to RBC transfusion burden (Malcovati et al., Haematologica 2006). Patients with chronic anemia despite frequent RBC transfusions may be at risk for associated morbidity (e.g., heart failure, falls, fatigue) and reduced quality of life (Crawford et al., Cancer 2002;95:888-895).

[0009] Hypomethylating agents (HMAs) (e.g., azacitidine and decitabine) are approved for the treatment of all French-American-British (FAB) subtypes of MDS, including some patients with lower-risk MDS. While these agents reduce the need for blood transfusions in patients with higher-risk MDS, there is no evidence of improved long-term outcomes for lower-risk patients who received HMAs after ESA failure. In a retrospective study of 1,698 patients with non-del(5q) lower-risk MDS treated with ESAs, patients who subsequently received HMAs (n=194) after ESA failure did not experience a significant improvement in 5-year overall survival (Park et al., J Clin Oncol. 2017;35(14):1591-1597). Other reports suggest that in cohorts of lower-risk MDS patients who are transfusion-dependent after ESA failure, azacitidine induces RBC transfusion independence ("RBC-TI") in approximately 14%–33% of patients (Fili et al., Clin Cancer Res. 2013;19:3297–3308; Thepot et al., Haematologica. 2016;101:918–925; Tobiasson et al., Blood Cancer J. 2014:4,e189). Given the limited benefit and observed toxicities (neutropenia, infection), azacitidine cannot be recommended as treatment for these patients (Tobiasson et al., Blood Cancer J. 2014).

[0010] The del(5q) chromosomal abnormality is observed in 10%–15% of patients with MDS and is associated with a favorable prognosis (Oliva et al., Ann Hematol. 2013;92(1):25-32). Treatment with lenalidomide results in transfusion independence in approximately two-thirds of such patients (Ades et al., Lancet 2014; Fenaux et al., Blood 2013;121(21):4280-4286). In a phase 3 trial, the median duration of transfusion independence (TI) was not reached (median follow-up, 1.55 years) (Fenaux et al., Blood 2011;118(14):3765-3776). Myelosuppression is the most frequently reported grade 3 or 4 toxicity, and close monitoring of blood counts is required during the first few weeks of lenalidomide therapy (ibid.).

[0011] Lenalidomide has also been studied as a treatment for transfusion-dependent non-del(5q) MDS, which accounts for 85%–90% of the MDS population. The majority of these patients do not respond to lenalidomide. Hematologic toxicities (i.e., neutropenia and thrombocytopenia) are milder than those observed in patients with del(5q) MDS (Loiseau et al., Exp Hematol. 2015;43(8):661–72). Similar to HMAs, treatment with lenalidomide after ESA failure has not been shown to significantly improve overall survival when used to treat lower-risk patients with non-del(5q) MDS (Park et al., J Clin Oncol. 2017). Immunosuppressive therapy is a treatment option for certain lower-risk non-del(5q) patients, but no significant effect on disease-free survival has been observed, and adverse events, including hematologic toxicity and associated serious adverse events such as bleeding and infection, have been reported (Almeida et al., Leukemia Res. 2017). Allogeneic stem cell transplantation is typically reserved for medically fit higher-risk MDS patients, but may be considered an option for select lower-risk patients, such as those under 60 to 70 years of age with IPSS intermediate-1 risk MDS, poor-risk cytogenetics, or persistent elevated blast counts, when alternative treatment options are ineffective (ibid.). [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Cheson et al.,Blood 2006;108:419-425 [Non-patent document 2] Vardiman,et al.,Blood 2009;114(5):937-951 [Non-patent document 3] Greenberg et al.,Blood 1997;89(6):2079-2088 [Non-patent document 4] Almeida et al.,Leukemia Res.2017;52:50-57 [Non-patent document 5] Malcovati et al.,J Clin Oncol 2005;23:7594-7603 [Non-patent document 6] Malcovati et al., Haematologica 2006;91:1588-1590 [Non-Patent Document 7] Steensma Dp.,Mayo Clinic Proc.2015;90(7):969-983 [Non-patent document 8] Ades et al.,Lancet 2014;383(9936):2239-2252

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[0013] Methods for treating MDS in a subject and methods for monitoring treatment are provided. Methods for identifying MDS subjects suitable for treatment with a telomerase inhibitor and methods for treating MDS are also provided. The subject methods can include administering an effective amount of a telomerase inhibitor to a subject and evaluating the variant allele frequency (VAF) in a biological sample obtained from the subject for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1. In some cases, a reduction of 25% or more in the VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 identifies a subject who is likely to benefit from treatment with a telomerase inhibitor. The subject methods can include administering an effective amount of a telomerase inhibitor to a subject and evaluating the VAF in a biological sample obtained from the subject for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1. In some cases, the VAF of one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 is reduced by 25% or more, thereby identifying subjects who are likely to benefit from treatment with a telomerase inhibitor.The subject can be in a state where they have not been treated with HMA, lenalidomide, or both.In some cases, the subject is classified as having IPSS low-risk or intermediate-1-risk MDS and / or MDS that is relapsed or refractory to ESA.In certain cases, the subject is non-del5q.In some examples, the telomerase inhibitor is imetelstat or imetelstat sodium.

[0014] Provided is a method for treating MDS in a subject with a telomerase inhibitor.In particular, the treatment with the telomerase inhibitor is continued if the VAF of one or more of the following genes is observed to be reduced by 25% or more after the administration of the telomerase inhibitor in the subject.In particular, the treatment with the telomerase inhibitor is continued if the VAF of one or more of the following genes is observed to be reduced by 25% or more after the administration of the telomerase inhibitor in the subject.The telomerase inhibitor can be imetelstat or imetelstat sodium.The subject can be in a state where they have not been treated with HMA, lenalidomide, or both.In some cases, the subject is classified as having IPSS low-risk or intermediate-1 risk MDS and / or MDS that is relapsed or refractory to ESA.In certain cases, the subject is non-del5q. In some cases, the subject is classified as having an intermediate IPSS and cytogenetic poor risk.

[0015] In some cases, a method for treating MDS in a subject with a telomerase inhibitor such as imetelstat includes administering a telomerase inhibitor such as imetelstat to the subject, and continuing to administer a telomerase inhibitor such as imetelstat to the subject because a 25% or greater reduction in VAF has been observed for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 after administering a telomerase inhibitor such as imetelstat. The telomerase inhibitor can be imetelstat or imetelstat sodium. The subject can be in a state where they have not been treated with HMA, lenalidomide, or both. In some cases, the subject is classified as having MDS with IPSS low risk or intermediate 1 risk and / or MDS that is relapsed or refractory to ESA. In certain cases, the subject is non-del5q. In some cases, the subject is classified as having IPSS intermediate and cytogenetic poor risk.

[0016] In some cases, a method for treating MDS in a subject with a telomerase inhibitor such as imetelstat includes administering a telomerase inhibitor such as imetelstat to the subject, and continuing to administer a telomerase inhibitor such as imetelstat to the subject because a 25% or greater reduction in VAF has been observed for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 after administering a telomerase inhibitor such as imetelstat. The telomerase inhibitor can be imetelstat or imetelstat sodium. The subject can be naive to treatment with HMA, lenalidomide, or both. In some cases, the subject is classified as having MDS with IPSS low risk or intermediate 1 risk and / or MDS that is relapsed or refractory to ESA. In certain cases, the subject is non-del5q. In some cases, the subject is classified as having IPSS intermediate and cytogenetic poor risk.

[0017] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments of the invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown. [Brief explanation of the drawings]

[0018] [Figure 1]Study design for a Phase 3 trial of imetelstat to treat MDS. *Received at least 8 weeks of ESA therapy (≥40,000 U epoetin alfa, ≥30,000 U epoetin beta, or 150 mcg or equivalent darbepoetin alfa per week) but without an increase in Hgb of ≥1.5 g / dL from ≥8 weeks of ESA therapy, or a reduction in RBC transfusion requirement or transfusion dependency of ≥4 units / 8 weeks, or a reduction in Hgb of ≥1.5 g / dL after hematologic improvement. **Percentage of patients who have not received any RBC transfusions for at least 8 consecutive weeks from study initiation (8-week TI); Percentage of patients who have not received any RBC transfusions for at least 24 consecutive weeks from study initiation (24-week TI). (EPO = erythropoietin, ESA = erythropoietin-stimulating agent, G-CSF = granulocyte colony-stimulating factor, Hgb = hemoglobin, IPSS = International Prognostic Scoring System, ITT = intention to treat, RBC = red blood cells, HI-E = hematologic improvement-red blood cells, HMA = hypomethylating agent, MDS = myelodysplastic syndrome.) [Figure 2] Uninterrupted, sustained TI with imetelstat treatment. *Pretreatment hemoglobin was 6.2 g / dL and transfusion burden was 5 units / 8 weeks prior to study initiation. Hemoglobin during the study was <6.5 g / dL for the majority of the TI period, but no transfusions were given. RBC = red blood cells, TI = transfusion independence. [Figure 3] Highly statistically significant and clinically meaningful durability of TI. *Kaplan-Meier estimates of duration of RBC TI; 8-week TI responder analysis set; hazard ratios are from Cox proportional hazards models stratified by prior RBC transfusion burden (≤6 units RBC vs. >6 units RBC) and IPSS risk group (low vs. intermediate), with treatment as the only covariate. P values ​​for superiority of imetelstat versus placebo in hazard ratios are based on stratified log-rank tests. CI = confidence interval, HR = hazard ratio, RBC = red blood cells, TI = transfusion independence. [Figure 4A]Transfusion independence rates assessed over time. Imetelstat demonstrated increased magnitude of benefit compared with placebo at longer TI. P values ​​are based on Cochran Mantel-Haenszel tests stratified for previous RBC transfusion burden (≤6 or >6 RBCs / 8 weeks) and baseline IPSS risk score (low risk or intermediate 1 risk). TI = transfusion independence. [Figure 4B] Rate of TI >1 year in imetelstat-treated patients versus placebo. [Figure 4C] Rates of TI >1 year in imetelstat-treated patients showing 8+ week and 24+ week TI responders. [Figure 5A] The mean change in hemoglobin levels over time demonstrates a highly statistically significant increase in hemoglobin levels after imetelstat treatment compared to placebo. The mean change from minimum Hgb to post-transfusion values ​​over the 14 days during the 8 weeks prior to the first day of dosing is shown. Data points with fewer than four patients are not shown. P values ​​are based on a repeated measures mixed model with an autoregressive moving average (ARMA(1,1)) covariance structure, with Hgb change as the dependent variable and week, stratification factors, minimum Hgb during the 8 weeks prior to the first day of dosing, and treatment arm as independent variables. Hgb = hemoglobin; SE = standard error; TI = transfusion independence. [Figure 5B] Mean change in central hemoglobin over time in 1+ year TI responders. For placebo patients, pretreatment hemoglobin was 6.2 g / dL and transfusion burden was 5 U / 8 weeks. During the study, hemoglobin remained below 6.5 g / dL for the majority of the TI period. BL = baseline. [Figure 6] The change in RBC transfusion units over time shows a statistically significant reduction in the number of RBC units transfused after imetelstat treatment compared to placebo. P values ​​are based on a repeated measures mixed model with an autoregressive moving average (ARMA(1,1)) covariance structure, with change in RBC transfusion as the dependent variable and week, stratification factors, previous transfusion burden, and treatment arm as independent variables. RBC = red blood cells; SE = standard error. [Figure 7A]Key subgroup analyses: 8-week TI by ring-sideroblast (RS) status, previous transfusion burden, and IPSS risk category. [Figure 7B] Key subgroup analyses. 24-week TI by RS status, previous transfusion burden, and IPSS risk category. A forest plot of the primary endpoint according to subgroup is shown, which is the percentage difference for imetelstat compared with placebo in 8-week TI rate between the indicated subgroups. 95% CIs are based on the Wilson Score method. P values ​​are based on Cochran-Mantel-Haenszel, controlling for previous RBC transfusion burden (4 to 6 units vs. >6 units RBCs) and International Prognostic Scoring System (IPSS) risk group (low vs. intermediate 1) applicable at randomization. A statistically significant 8-week TI rate (p<0.05) and a similar degree of clinical benefit were observed with imetelstat across all subgroups. *Cochran-Mantel-Haenszel test stratified for previous RBC transfusion burden (<6 units or >6 units RBCs / 8 weeks) and baseline IPSS risk score (low risk or intermediate 1 risk). CI = confidence interval; IPSS = International Prognostic Scoring System; IWG = International Working Group; RBC = red blood cells; RS+ = ring sideroblasts positive; RS- = ring sideroblasts negative. [Figure 7C] Key subgroup analyses. (C) Baseline TA, TL, and hTERT levels, and (D) 8-week TI by baseline TA, TL, and hTERT levels. (C) and (D) show forest plots of TI response according to subgroup, which are the percentage differences in imetelstat compared to placebo in 8-week and 24-week TI rates between the indicated subgroups. Unfavorable prognostic mutations were defined as the presence of TP53, EZH2, ETV6, RUNX1, or ASXL1, based on Bejar et al. N Engl J Med 2011. [Figure 7D]Key subgroup analyses. (C) Baseline TA, TL, and hTERT levels, and (D) 8-week TI by baseline TA, TL, and hTERT levels. (C) and (D) show forest plots of TI response according to subgroup, which are the percentage differences in imetelstat compared to placebo in 8-week and 24-week TI rates between the indicated subgroups. Unfavorable prognostic mutations were defined as the presence of TP53, EZH2, ETV6, RUNX1, or ASXL1, based on Bejar et al. N Engl J Med 2011. [Figure 8] TI for patients with and without RS. The percentage of patients with the indicated RS status in each study group who had a TI of 24 weeks or longer is shown. For the key secondary endpoint of TI of 24 weeks or longer, there was a difference of 28% (P=0.003) for patients with RS and 21% (P=0.019) for patients without RS for imetelstat compared with placebo. Patients without RS who received placebo did not achieve a 24-week TI. Error bars represent 95% confidence intervals calculated using exact Clopper-Pearson confidence intervals. NE indicates not estimable. The primary endpoint (8-week TI) and secondary endpoint (24-week TI) by RS status show that both RS+ and RS- showed statistically significant improvements in both 8-week and 24-week TI. *Kaplan-Meier estimates of duration of RBC TI, 8-week / 24-week TI responder analysis set. P-values ​​for TI rate are based on Cochran-Mantel-Haenszel tests stratified for previous RBC transfusion burden (≤6 units or >6 units RBC / 8 weeks) and baseline IPSS risk score (low risk or intermediate-1 risk); P-values ​​for TI duration are based on stratified log-rank tests. CI = confidence interval, NE = not evaluable, RS+ = ring sideroblast positive, RS- = ring sideroblast negative, TI = transfusion independent. [Figure 9]Nucleator data in imetelstat-treated patients show a greater than 50% reduction in VAF for SF3B1, TET2, DNMT3A, and ASXL1 mutations. The ratios below the bars represent the number of patients with a ≥50% reduction in VAF as the numerator and the total number of patients with detectable baseline assessment (≥5% VAF) for the identified mutation and any post-baseline mutation assessment as the denominator. *P values ​​are based on a Cochran-Mantel-Haenszel test stratified for previous RBC transfusion burden (≤6 units or >6 units RBCs / 8 weeks) and baseline IPSS risk score (low risk or intermediate-1 risk). [Figure 10] Association of maximum reduction in SF3B1 or TET2 VAF with TI response (at least 8 weeks, 24 weeks, 1 year, and no response) between imetelstat and placebo treatment in patients with a baseline VAF ≥ 5% and at least one post-baseline mutation assessment. [Figure 11] Significant TI in patients treated with imetelstat compared with placebo. P values ​​are based on Cochran-Mantel-Haenszel, controlling for previous RBC transfusion burden (≤6 units vs. ≥6 units RBCs) and IPSS risk group (low vs. intermediate), applicable at randomization. [Figure 12A] Reductions in VAF for SF3B1, TET2, DNMT3A, and ASXL1 mutations correlate with longer duration of TI and increased hemoglobin. Comparisons between study arms are shown for maximum percentage change from baseline during treatment in mutant VAF for the indicated genes. For three of four genes frequently mutated in MDS, VAF reductions were significantly greater in patients treated with imetelstat than in patients treated with placebo: SF3B1 (P<0.001), TET2 (P=0.032), DNMT3A (P=0.019), and ASXL1 (P=NS). [Figure 12B]Correlation of SF3B1, TET2, DNMT3A, and ASXL1 mutations with reduction in VAF and longer duration of TI and increased hemoglobin. Figure 1 shows the correlation between the maximum percentage reduction from baseline in SF3B1 VAF with imetelstat treatment and the duration of transfusion independence in the imetelstat group. Results show a greater reduction in SF3B1 VAF and longer duration of TI (n=78, Pearson's r=-0.549, P<0.001). [Figure 12C] Correlation of SF3B1, TET2, DNMT3A, and ASXL1 mutations with reductions in VAF and longer duration of TI and increased hemoglobin. Correlation between maximum percentage reduction from baseline in SF3B1 VAF and maximum change from pretreatment hemoglobin levels in the imetelstat group is shown. Results demonstrate greater reductions in SF3B1 VAF and higher increases in hemoglobin (n=67, Pearson's r=-0.626, P<0.001). Hemoglobin measurements from blood drawn within 14 days of transfusion were excluded. Mutation analysis included patients with ≥5% detectable variant alleles at baseline and at least one post-baseline mutation assessment. [Figure 13A] Time to first sustained and meaningful improvement in fatigue, and the association between TI and HI-E response and sustained and meaningful improvement in fatigue. Sustained improvement in fatigue in imetelstat-treated patients. Kaplan Meier survival estimates of time to first sustained improvement for the FACIT fatigue scale, PRO population. [Figure 13B]Time to first sustained and meaningful improvement in fatigue, and the association of TI and HI-E response with sustained and meaningful improvement in fatigue. Sustained improvement in fatigue in imetelstat-treated patients. Graph showing the percentage of subjects with sustained and meaningful improvement in fatigue for imetelstat responders, imetelstat non-responders, placebo responders, and placebo non-responders with transfusion independence (TI) and HI-E according to IWG 2006 criteria for 8 weeks or more or 24 weeks or more. A sustained and meaningful improvement in fatigue was defined as an increase of at least 3 points in the FACIT Fatigue score for at least two consecutive, non-missed cycles. Patients for whom fatigue data were available at baseline were included in the analysis. [Figure 14] IMerge Phase 3: Study design and patient disposition. [Figure 15] Swimmer plot of TI interval in patients with a TI of 8 weeks or more. [Figure 16] Relative dose intensity per cycle (%). Relative dose intensity is shown for a starting dose of 7.5 mg / kg. [Figure 17A] Correlation of TET2 and DNMT3A mutations with reduced VAF and longer duration of TI and increased hemoglobin. Correlation between the maximum percentage reduction from baseline in TET2 VAF with imetelstat treatment and the duration of transfusion independence in the imetelstat group is shown. Results show a greater reduction in TET2 VAF with longer duration of TI (n=38, Pearson's r=-0.577, P<0.001). [Figure 17B]Correlation of TET2 and DNMT3A mutations with VAF reduction and longer duration of TI and hemoglobin increase. Correlation between maximum percentage reduction from baseline in DNMT3A VAF with imetelstat treatment and duration of transfusion independence in the imetelstat group is shown. Duration of RBC TI fitted to maximum percentage reduction from baseline in DNMT3A VAF in the imetelstat arm. The fitted line and p-value are based on linear regression with duration of RBC TI as the dependent variable and maximum percentage reduction from baseline in DNMT3A VAF as the independent variable. Correlation coefficients are based on Pearson correlation. N represents imetelstat arm subjects in the mutation biomarker analysis set who had a detectable score (≥5) in DNMT3A at baseline and any post-baseline mutation assessment. Results show a greater reduction in DNMT3A VAF and a longer TI duration (n=18, Pearson's r=-0.545, P<0.019). [Figure 17C] Correlation of TET2 and DNMT3A mutations with reduced VAF and longer duration of TI and increased hemoglobin. Correlation between maximum percentage reduction from baseline in TET2 VAF and maximum change from pretreatment hemoglobin levels in the imetelstat group is shown. Results demonstrate greater reductions in TET2 VAF and higher increases in hemoglobin (n=32, Pearson's r=-0.659, P<0.001). Hemoglobin measurements from blood drawn within 14 days of transfusion were excluded. Mutation analysis included patients with ≥5% detectable variant alleles at baseline and at least one post-baseline mutation assessment. [Figure 17D]Correlation of TET2 and DNMT3A mutations with reduced VAF and longer duration of TI and increased hemoglobin. Correlation between the maximum percentage reduction from baseline in DNMT3A VAF and the maximum change from pretreatment hemoglobin levels in the imetelstat group is shown. Results demonstrate greater reductions in DNMT3A VAF and higher increases in hemoglobin (n=13, Pearson's r=-0.667, P<0.013). Hemoglobin measurements from blood drawn within 14 days of transfusion were excluded. Mutation analysis included patients with ≥5% detectable variant alleles at baseline and at least one post-baseline mutation assessment. [Figure 18] Imetelstat treatment resulted in a sustained reduction in SF3B1 VAF over time. [Figure 19A] RBC-TI at weeks 8 (19A) and 24 (19B) correlated with reductions in RS+ cells, cytogenetic response, and reductions in VAF in patients treated with imetelstat. P values ​​were calculated using Fisher's exact test for "yes" and "no" for each outcome. BM, bone marrow; CR, complete response; IRC, independent review committee; PR, partial response. [Figure 19B] RBC-TI at weeks 8 (19A) and 24 (19B) correlated with reductions in RS+ cells, cytogenetic response, and reductions in VAF in patients treated with imetelstat. P values ​​were calculated using Fisher's exact test for "yes" and "no" for each outcome. BM, bone marrow; CR, complete response; IRC, independent review committee; PR, partial response. [Figure 20] A higher percentage of patients treated with imetelstat versus placebo had a 50% or greater reduction in central bone marrow RS, and RBC-TI responders were higher in patients who achieved a 50% or greater reduction in central bone marrow RS. [Figure 21]Swimmer plots of 22 LR-MDS patients (21 imetelstat-treated patients and 1 placebo-treated patient) who achieved ≥1 year of RBC TI. Patients taking placebo: Pre-treatment Hb level was 6.2 g / dL and transfusion burden was 5 U per 8 weeks before study initiation, while during the study, Hb levels remained below 6.2 g / dL for the majority of the TI period and no transfusions were given. PBO: Placebo. [Figure 22] Summary of TI response rates by baseline mutation status of the most frequently mutated genes. [Figure 23] Heatmap of mutational burden changes in 18 imetelstat-treated patients with TI of ≥1 year. [Figure 24] TI rates according to the number of baseline mutations are shown. [Figure 25] Summary of TI response rates by baseline mutation status of four sets of genes involved in different biological functions. [Figure 26A] Spaghetti plot of mutational VAF change over time in imetelstat-treated patients. Different patterns of VAF reduction over time for different genes during imetelstat treatment are shown. The 4.7 mg / kg dose represents an actual dose of 4.2 to 5.35 mg / kg. The 6.0 mg / kg dose represents an actual dose of 5.35 to 6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75 to 8.45 mg / kg. Include data from scheduled and unscheduled visits. [Figure 26B] Spaghetti plot of mutational VAF change over time in imetelstat-treated patients. Different patterns of VAF reduction over time for different genes during imetelstat treatment are shown. The 4.7 mg / kg dose represents an actual dose of 4.2 to 5.35 mg / kg. The 6.0 mg / kg dose represents an actual dose of 5.35 to 6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75 to 8.45 mg / kg. Include data from scheduled and unscheduled visits. [Figure 26C]Spaghetti plot of mutational VAF change over time in imetelstat-treated patients. Different patterns of VAF reduction over time for different genes during imetelstat treatment are shown. The 4.7 mg / kg dose represents an actual dose of 4.2 to 5.35 mg / kg. The 6.0 mg / kg dose represents an actual dose of 5.35 to 6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75 to 8.45 mg / kg. Include data from scheduled and unscheduled visits. [Figure 27A] Limited reduction in VAF of certain genes over time in placebo-treated patients. The 6.0 mg / kg dose represents an actual dose of 5.35-6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75-8.45 mg / kg. The 9.4 mg / kg dose represents an actual dose of 8.45-11 mg / kg. Include data from scheduled and unscheduled visits. [Figure 27B] Limited reduction in VAF of certain genes over time in placebo-treated patients. The 6.0 mg / kg dose represents an actual dose of 5.35-6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75-8.45 mg / kg. The 9.4 mg / kg dose represents an actual dose of 8.45-11 mg / kg. Include data from scheduled and unscheduled visits. [Figure 27C] Limited reduction in VAF of certain genes over time in placebo-treated patients. The 6.0 mg / kg dose represents an actual dose of 5.35-6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75-8.45 mg / kg. The 9.4 mg / kg dose represents an actual dose of 8.45-11 mg / kg. Include data from scheduled and unscheduled visits. [Figure 27D]Limited reduction in VAF of certain genes over time in placebo-treated patients. The 6.0 mg / kg dose represents an actual dose of 5.35-6.75 mg / kg. The 7.5 mg / kg dose represents an actual dose of 6.75-8.45 mg / kg. The 9.4 mg / kg dose represents an actual dose of 8.45-11 mg / kg. Include data from scheduled and unscheduled visits. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present application provides methods for treating and monitoring the effectiveness of treatment in subjects with MDS. Also provided is a method for identifying subjects with MDS suitable for treatment with a telomerase inhibitor. The subject method can include administering an effective amount of a telomerase inhibitor to a subject and assessing the VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject. In some cases, a 25% or greater reduction in the VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 identifies subjects likely to benefit from treatment with a telomerase inhibitor. The subject method can include administering an effective amount of a telomerase inhibitor to a subject and assessing the VAF for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject. In some cases, a 25% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 identifies subjects who are likely to benefit from treatment with a telomerase inhibitor.

[0020] In some cases, treatment with a telomerase inhibitor such as imetelstat is continued if a 25% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 is observed in the subject. Thus, in some cases, a method of treating MDS in a subject with a telomerase inhibitor such as imetelstat includes administering a telomerase inhibitor such as imetelstat to the subject, and continuing to administer the telomerase inhibitor such as imetelstat to the subject since a 25% or greater reduction in VAF has been observed for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 after administration of the telomerase inhibitor such as imetelstat. The reduction in VAF may be a 10% or more, or 15% or more, or 20% or more, or 25%, for example, a 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 100% reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1.

[0021] In some cases, treatment with a telomerase inhibitor such as imetelstat is continued if a 25% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 is observed in the subject. Thus, in some cases, a method of treating MDS in a subject with a telomerase inhibitor such as imetelstat includes administering a telomerase inhibitor such as imetelstat to the subject, and continuing to administer the telomerase inhibitor such as imetelstat to the subject since a 25% or greater reduction in VAF has been observed for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 after administration of the telomerase inhibitor such as imetelstat. The reduction in VAF may be a reduction of 10% or more, or 15% or more, or 20% or more, or 25% or more, such as 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 100% in VAF for one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1.

[0022] Treatment with a telomerase inhibitor may be discontinued if a 25% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 is not observed in the subject after administration of the telomerase inhibitor and additional factors suggests discontinuation of treatment. Treatment with a telomerase inhibitor may be discontinued if a 25% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1 is not observed in the subject after administration of the telomerase inhibitor and additional factors suggests discontinuation of treatment. For example, treatment with a telomerase inhibitor may be discontinued if TI is not achieved and / or adverse side effects are observed.

[0023] The subject may be naive to treatment with HMA, lenalidomide, or both. In some cases, the treated subject is classified as having IPSS low-risk MDS, IPSS intermediate-1 risk MDS, MDS that has relapsed to ESA, MDS that is refractory to MS, or a combination thereof. The subject may also be non-del5q. For clarity, but not limitation, of the disclosure, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or uses of the invention. In some embodiments, the subject is diagnosed with trisomy 8.

[0024] A.Definition SF3B1 (splice factor 3b subunit 1) is the largest subunit of the SF3B complex and functions by serving as a core component of the U2 snRNP, which is important for branch site recognition and the early steps of spliceosome assembly. SF3B1 hotspot mutations may be selected from E622D, R625C / L / G, H662Q / N / D / Y, T663P, K666R / T / Q / N, K700E, A744P, and / or E783K.

[0025] The protein encoded by this gene, TET2 (Tet methylcytosine dioxygenase 2), is a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine.

[0026] The ASXL1 (additional sex combs like 1) gene encodes a polycomb chromatin-binding protein and is involved in the epigenetic regulation of gene expression.

[0027] The DNMT3A (DNA methyltransferase 3 alpha) gene encodes an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, a process called DNA methylation.

[0028] The CUX1 (cut like homeobox 1) gene encodes a tumor suppressor.

[0029] As used herein, the term "about" when referring to a measurable value, such as an amount, temporal duration, etc., is meant to encompass a variation of ±20% to ±0.1%, preferably ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for performing the disclosed methods.

[0030] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that is acceptable for a given dosage regimen and safe for the mammal). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, which salt is derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, and, when the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride. Pharmaceutically acceptable salts of interest include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, cholinate, ethylenediamine, lysine, lithium, magnesium, meglumine, procaine, potassium, sodium, tromethamine, N-methylglucamine, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, zinc, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine salts.

[0031] The term "salt(s) thereof" refers to a compound formed when an acid proton is replaced with a cation, such as a metal cation or an organic cation. Preferably, the salt is a pharmaceutically acceptable salt. By way of example, salts of the present compounds include those in which the compound is protonated with an inorganic or organic acid to form a cation, using the conjugate base of the inorganic or organic acid as the anionic component of the salt. Salts of interest include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, cesium, cholinate, ethylenediamine, lithium, magnesium, meglumine, procaine, N-methylglucamine, piperazine, potassium, sodium, tromethamine, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine salts. It should be understood that for any of the oligonucleotide structures depicted herein, including a backbone of internucleoside linkages, such oligonucleotides may include any convenient salt form. In some embodiments, the acidic form of the internucleoside bond is shown for simplicity. In certain instances, the salt of the subject compound is a monovalent cation salt. In certain cases, the salt of the subject compound is a divalent cation salt. In certain instances, the salt of the subject compound is a trivalent cation salt. A "solvate" refers to a complex formed by the combination of a solvent molecule with a solute molecule or ion. The solvent may 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.

[0032] "Stereoisomer" and "stereoisomer" refer to compounds that have the same atomic connectivity but different atomic arrangements in space. Stereoisomers include, for example, cis-trans isomers, E and Z isomers, enantiomers, and diastereomers. With respect to any group disclosed herein that contains one or more substituents, it is understood that such group does not include any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. All stereoisomers are intended to be included within the scope of the present disclosure.

[0033] Those skilled in the art will recognize that other tautomeric configurations of the groups described herein are possible. It is understood that all tautomeric forms of the subject compounds are encompassed by the depicted structures, even if not specifically shown, where one possible tautomeric configuration of the groups of the compounds is encompassed by the depicted structures.

[0034] Pharmaceutically acceptable salt solvates of the stereoisomeric tautomers of the subject compounds are intended to be included and are intended to be included within the scope of this disclosure.

[0035] Before describing certain embodiments in more detail, it is to be understood that this invention is not limited to certain described embodiments, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0036] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in the stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0038] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with the cited publications. The citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0039] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," or a "negative" limitation in connection with the recitation of claim elements.

[0040] Each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0041] As used throughout, "MDS" refers to myelodysplastic syndrome or multiple myelodysplastic syndromes.

[0042] The term "one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1" refers to these genes alone or in any combination of two, three, or four of these genes. One skilled in the art can readily discern that six combinations of any two of these genes can be made, four combinations of any three of these genes can be made, and one combination of all of these genes can be made. The term "one or more of the genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1" refers to these genes alone or in any combination of two, three, four, or five of these genes. One skilled in the art can readily discern that ten combinations of any two of these genes can be made, ten combinations of any three of these genes can be made, five combinations of any four of these genes can be made, and one combination of all of these genes can be made.

[0043] Furthermore, one or more genes can be selected from the subset of genes SF3B1, TET2, DNMT3A, and ASXL1. One or more genes can be selected from the subset of genes SF3B1, TET2, DNMT3A, CUX1, and ASXL1. For example, one or more genes can be selected from genes TET2, DNMT3A, and ASXL1. One or more genes can also be selected from the group of genes SF3B1, TET2, and DNMT3A. Alternatively, one or more genes can be selected from genes SF3B1, DNMT3A, and ASXL1. Still further, one or more genes can be selected from genes SF3B1, TET2, and ASXL1. Furthermore, one or more genes can be selected from SF3B1, TET2, and CUX1, or from SF3B1, DNMT3A, and CUX1, or from SF3B1, TET2, and CUX1, or from TET2, DNMT3A, and CUX1. Furthermore, the one or more genes are selected from any one of the following groups: genes SF3B1 and TET2, SF3B1 and DNMT3A, SF3B1 and ASXL1, TET2 and DNMT3A, TET2 and ASXL1, DNMT3A and ASXL1, SF3B1 and CUX1, TET2 and CUX1, DNMT3A and CUX1, ASXL1 and CUX1. In some cases, the VAF for one gene is analyzed from genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1.

[0044] The biological sample obtained from the subject may be a peripheral blood sample or a bone marrow sample, for example, cells derived from peripheral blood or bone marrow obtained from the subject.

[0045] It is within the scope of the present invention to use each of these combinations in the methods disclosed herein, and a comparison of VAF between two time points is performed for the same gene combination.

[0046] B. Pharmacodynamics (PD) The present disclosure is based in part on the pharmacodynamic effects that demonstrate the association between the response to telomerase inhibitor therapy, particularly imetelstat therapy, in subjects with MDS and the VAF of one or more of the following genes: SF3B1, TET2, DNMT3A, and ASXL1 by 25% or more.In some cases, subjects that exhibit a VAF of one or more of the following genes: SF3B1, TET2, DNMT3A, and ASXL1 by 25% or more typically show a TI response of at least 8 weeks.Conversely, subjects that do not show such a 25% or more reduction typically do not show a TI response of at least 8 weeks.

[0047] Thus, the present disclosure provides for the stratification and identification or selection of patients likely to benefit from telomerase inhibition therapy for MDS, and provides methods for monitoring response, relapse, and prognosis in subjects undergoing such treatment.

[0048] Aspects of the present disclosure include the method for identifying or selecting a subject with MDS for treatment with a telomerase inhibitor, and the method for treating MDS. Also provided is a method for monitoring the effectiveness of treatment in a subject with MDS. In some cases, the pharmacodynamic effect that the subject method embodiment is based on is the VAF of one or more of the following genes: SF3B1, TET2, DNMT3A, and ASXL1 is reduced by 25% or more.

[0049] In some cases, a 25% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1 is observed at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the first administration of a telomerase inhibitor, such as imetelstat. Such VAF measurements can be performed on cells derived from peripheral blood or bone marrow obtained from the patient. Peripheral blood or bone marrow samples can be obtained periodically from the patient, for example, every 4 to 6 weeks. In some cases, peripheral blood or bone marrow samples can be obtained from the patient each time the telomerase inhibitor is administered to the patient. Typically, to monitor telomerase inhibitor therapy or to determine whether to continue or discontinue telomerase inhibitor therapy, the VAF of the target gene can be measured before, particularly immediately before, for example, less than about 4 to 6 weeks before, the first dose of the telomerase inhibitor, such as imetelstat. The VAF of the target gene can be measured at 4-16 week intervals after administration of the telomerase inhibitor, for example, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 16 weeks. For uninterrupted monitoring of therapy, the VAF can be measured repeatedly after each such interval, for example, every 4 weeks, 6 weeks, 8 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 16 weeks.

[0050] The VAF of a target gene measured before, particularly shortly before, e.g., less than about 4-6 weeks before, administration of the first dose of a telomerase inhibitor, e.g., imetelstat, is referred to as the "baseline VAF."

[0051] The VAF of target gene can be determined and / or measured by any convenient method.One such method is to use whole blood collected in EDTA tube, and perform next-generation sequencing (NGS), for example, using LeukoVantage®.NGS using any other sequencing platform can be used, for example, nanopore sequencing, paired-end sequencing, Ion Torrent sequencing, and single molecule real-time (SMRT) sequencing.MDS gene panel can be used.For example, a gene panel including one or more of SF3B1, TET2, DNMT3A, and ASXL1 can be used. In a specific embodiment, a gene panel containing the following 36 genes was used: ASXL1, ATM, BCOR, CBL, CEBPA, CSF3R, CUX1, DNMT3A, ETNK1, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, IKZF1, JAK2, KRAS, NF1, NPM1, NRAS, PHF6, PTEN, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, STAT3, STK11, TET2, TP53, U2AF1, WT1, and ZRSR2. The NGS assay can be performed using a DNA bait capture method on the NextSeq® (Illumina®) platform. After DNA extraction from white blood cells, a targeted amplicon-based NGS method is used to detect mutations. The lower limit of sensitivity is 5%, which is superior to that of conventional Sanger DNA sequencing. The percentage of variant reads is reported and can be used to assess the size of the clonal population. VAF is the percentage of observed sequence reads that match a particular DNA variant divided by the overall coverage at that locus. Because NGS provides a nearly random sample, VAF is therefore a surrogate measure of the proportion of DNA molecules in the original specimen that carry the variant.

[0052] As mentioned above, the reduction of VAF means the reduction of malignant cell burden.For example, the VAF of one or more of the following genes SF3B1, TET2, DNMT3A, ASXL1 and CUX1 is reduced by 10% or more, or 15% or more, or 20% or more, or 25% or more, for example, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 100% or more, indicates that the subject is likely to benefit from the treatment with telomerase inhibitor.

[0053] In some cases, the reduction of VAF means that the duration of TI of the subject is longer.For example, the reduction of VAF can be correlated with the longer duration of TI of the subject compared with the subject that does not have the same reduction of VAF.In some cases, the reduction of VAF means that the time to TI occurrence in the subject is shorter.For example, the reduction of VAF can be correlated with the shorter time to TI occurrence in the subject compared with the subject that does not have the same reduction of VAF.

[0054] C.Treatment In some cases, the present disclosure describes methods of treating MDS with a telomerase inhibitor in subjects who have not received treatment with certain drugs, such as drugs selected from hypomethylating agents (HMAs) and lenalidomide. A subject is considered "naive" if they have not received a specific treatment for a disease. Treating MDS patients who are relapsed or refractory to ESA therapy with imetelstat can improve outcomes, including reducing the incidence of anemia.

[0055] The subject is a mammal that needs to be treated with MDS.Generally, the subject is a human patient.In some embodiments of the present invention, the subject can be a non-human mammal such as a non-human primate, an animal model (for example, an animal such as a rat that is used for drug screening, characterization and evaluation), and other mammals.As used herein, the terms patient and subject are used interchangeably.

[0056] As used herein, as is well known in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results.For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, the alleviation or improvement of one or more symptoms, whether detectable or undetectable, the reduction in the extent of disease, the stabilization (i.e., non-worsening) state of disease, the prevention of disease spread, the delay or slowing of disease progression, the improvement or alleviation of disease state, and remission (whether partial or total)."Treatment" can also mean the prolongation of survival compared to the expected survival if not receiving treatment.

[0057] In certain instances, the subject methods provide an enhanced therapeutic response in subjects not previously treated with a hypomethylating agent (HMA) or lenalidomide, compared to subjects previously so treated. "Enhanced therapeutic response" refers to a statistically significant improvement in the primary and / or secondary endpoints of an MDS therapy, and / or an improvement in one or more symptoms of MDS (e.g., as described herein), e.g., the rate and / or duration of red blood cell (RBC) TI, or the rate of hematological improvement (HI), compared to an appropriate control. In some instances, the subject methods provide a therapeutic effect of RBC TI, which lasts, for example, 4 weeks or longer, e.g., 5 weeks or longer, 6 weeks or longer, 7 weeks or longer, 8 weeks or longer, 9 weeks or longer, 10 weeks or longer, 12 weeks or longer, 16 weeks or longer, 20 weeks or longer, 24 weeks or longer, or even longer. In some instances, the time to TI and / or the duration of TI are significantly improved. In certain instances, the subject methods provide a TI duration of 24 weeks or more, eg, 30 weeks or more, 36 weeks or more, 42 weeks or more, 48 weeks or more, 60 weeks or more, or more.

[0058] Methylation inhibitors (HMAs) are drugs that inhibit DNA methylation, for example, by blocking the activity of DNA methyltransferase (DNA methyltransferase inhibitors / DNMT inhibitors). HMAs of interest include, but are not limited to, decitabine (CAS Registry Number: 2353-33-5, 5-aza-2'-deoxycytidine), azacitidine (CAS Registry Number: 320-67-2, 5-azacytidine), and guadecitabine (SGI-110). In some examples, the subject has not been treated with decitabine. In some examples, the subject has not been treated with azacitidine. In other examples, the subject has not been treated with both decitabine and azacitidine.

[0059] Lenalidomide is a drug used to treat various inflammatory diseases and cancers, including multiple myeloma and MDS. Lenalidomide (CAS Registry Number: 191732-72-6, 2,6-piperidinedione, 3-(4-amino-1,3-dihydro-1-oxo-2H-isoindol-2-yl)-), 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione) is a derivative of thalidomide. Lenalidomide has a variety of mechanisms of action, providing a wide range of biological activity that can be utilized to treat various blood cancers and solid cancers. In some instances, the subject is naive to lenalidomide treatment.

[0060] Deletion 5q (del5q) refers to a chromosomal abnormality found in certain forms of MDS subjects (Adema et al., Haematologica. 2013 Dec;98(12):1819-1821; Sole et al., Haematologica. 2005;90(9):1168-78). In some cases of the subject methods, the subject is a human patient with del5q. In some cases, the subject is a non-del5q human patient. A non-del5q subject is a subject who does not have a del5q chromosomal abnormality. In certain cases, the non-del5q subject is a human.

[0061] In certain instances, the subject has not been previously treated with either a hypomethylating agent (HMA) or lenalidomide, and does not have a del(5q) chromosomal abnormality (e.g., is non-del5q). In certain instances, the non-del5q subject is a human.

[0062] In certain embodiments, the subject is a human patient with cytogenetic intermediate risk or poor risk.In some examples, the subject is a human patient diagnosed with trisomy 8.In certain examples, the subject is a human patient with mosaic trisomy 8.In other examples, the subject is a human patient with non-mosaic trisomy 8.The term "mosaic" is used herein in its conventional sense to refer to a state in which cells in a subject have different genetic makeups.In a human patient diagnosed with mosaic trisomy 8, some of the subject's cells have three copies of chromosome 8, while other cells have two copies of chromosome 8.

[0063] In certain embodiments, a method includes identifying a subject with MDS for treatment with a telomerase inhibitor, such as imetelstat, the method including identifying a subject with trisomy 8 (mosaic or non-mosaic); measuring the VAF in one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1 in a biological sample obtained from the patient after administration of the telomerase inhibitor; and comparing the VAF in one or more of the SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes in the biological sample with a baseline VAF in one or more of the SF3B1, TET2, DNMT3A, and ASXL1 genes before administration of the telomerase inhibitor, wherein a reduction of 25% or more in the VAF for one or more of the SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes identifies a patient likely to benefit from treatment with the telomerase inhibitor.

[0064] In another embodiment, a method includes treating MDS in a subject diagnosed with trisomy 8, the method including identifying a subject diagnosed with trisomy 8, administering an effective amount of a telomerase inhibitor to the subject, and assessing VAF in one or more of the SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes in a biological sample obtained from the patient after administration of the telomerase inhibitor, such as imetelstat. The method then includes continuing to administer the telomerase inhibitor to the subject if a 25% or greater reduction in VAF for one or more of the SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes is observed.

[0065] In yet another embodiment, a method comprises monitoring the effectiveness of a treatment in a subject with MDS, the method comprising measuring VAF in one or more of SF3B1, TET2, DNMT3A, ASXL1, and CUX1 in a biological sample obtained from a patient diagnosed with trisomy 8 after administration of a telomerase inhibitor; and comparing the VAF in one or more of SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes in the biological sample with a baseline VAF in one or more of SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes before administration of the telomerase inhibitor, wherein a reduction of 25% or more in VAF for one or more of SF3B1, TET2, DNMT3A, ASXL1, and CUX1 genes identifies patients likely to benefit from treatment with the telomerase inhibitor.

[0066] In certain embodiments, the subject is a patient diagnosed with trisomy 8. In other embodiments, the subject is a patient diagnosed with trisomy 8 and has not been treated with a drug selected from HMA, lenalidomide, and combinations thereof. In other embodiments, the subject is a patient diagnosed with trisomy 8 and is a non-del5q human patient. In yet other embodiments, the subject is a patient diagnosed with trisomy 8, and MDS is relapsed or refractory MDS, for example, relapsed or refractory MDS to ESA.

[0067] In some cases, the present disclosure provides the use of a telomerase inhibitor, such as imetelstat, to treat a subject with MDS and / or monitor the effectiveness of treatment in a subject with MDS. Also provided is a method for identifying a subject with MDS suitable for treatment with a telomerase inhibitor. The use includes assessing the VAF for one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1 in a biological sample obtained from the subject after administering a telomerase inhibitor to the subject. In some cases, a reduction of 25% or more in the VAF for one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1 identifies a subject likely to benefit from treatment with a telomerase inhibitor.

[0068] In some cases, the use of telomerase inhibitors to treat MDS in a subject is continued when the VAF of one or more of the genes SF3B1, TET2, DNMT3A, ASXL1 and CUX1 is observed to be reduced by 25% or more in the subject.Telomerase inhibitor treatment can be discontinued if the VAF of one or more of the genes SF3B1, TET2, DNMT3A, ASXL1 and CUX1 is not reduced by 25% or more in the subject after the administration of telomerase inhibitors and additional factors suggests discontinuing treatment.For example, if TI is not achieved and / or adverse side effects are observed, telomerase inhibitor treatment can be discontinued. For example, treatment with a telomerase inhibitor may be discontinued if the VAF reduction for one or more of the genes SF3B1, TET2, DNMT3A, ASXL1, and CUX1 measured in a biological sample from a subject is 75% or less, or 70% or less, or 60% or less, or 55% or less, or 50% or less, or 45% or less, or 40% or less, or 35% or less, or 30% or less, or 25% or less, or 20% or less, or 15% or less, or 10% or less, or 5% or less.In such cases, a low reduction in VAF as described above may indicate that the subject is unlikely to benefit from treatment with a telomerase inhibitor.

[0069] The subject may be naive to treatment with HMA, lenalidomide, or both. In some cases, the subject is classified as having IPSS low-risk MDS, IPSS intermediate-1 risk MDS, MDS that has relapsed to ESA, MDS that is refractory to MS, or a combination thereof. The subject may also be non-del5q. For clarity, but not limitation, of the disclosure, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or uses of the invention. In some embodiments, the subject is diagnosed with trisomy 8.

[0070] D. Myelodysplastic syndrome (MDS) MDS is a group of diseases including cancers of the blood and bone marrow, which in some cases may be characterized by cytopenia resulting from ineffective hematopoiesis. The subject method can be used to treat various MDS, including, but not limited to, refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with unilineage dysplasia, chronic myelomonocytic leukemia, isolated del(5q) MDS, and unclassifiable MDS.

[0071] MDS is characterized by clonal myeloproliferation arising from malignant progenitor cell clones with shorter telomeres and multiple clonal genetic abnormalities. Expression of telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) is significantly increased in MDS and may play a role in dysregulated cell growth, leading to the continuous and uncontrolled proliferation of malignant progenitor cell clones. Higher TA and hTERT, as well as shorter telomere length, are poor prognostic features of lower-risk MDS patients, resulting in reduced overall survival. Lower-risk MDS patients who have relapsed after or are refractory to ESA therapy have limited treatment options for anemia. Targeting MDS clones with imetelstat can improve outcomes, including anemia, in MDS patients who have relapsed or are refractory to ESA therapy.

[0072] In some embodiments, the subject methods find use in alleviating at least one symptom associated with myelodysplastic syndromes, such as, for example, refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with unilineage dysplasia, and chronic myelomonocytic leukemia. In some embodiments, the symptoms include shortness of breath, fatigue, weakness, fainting, nosebleeds, bruising, bleeding from the mouth or gums, bloody stools, petechiae, or stroke.

[0073] In some instances, the subject has relapsed or refractory MDS. "Refractory MDS" refers to a patient who still has MDS cells in the bone marrow after treatment with any effective MDS-related therapy. "Relapsed MDS" refers to a patient whose MDS cells have returned to the bone marrow and whose normal blood cells have decreased after remission. In certain instances, the subject has MDS that is relapsed or refractory to ESA. ESAs increase hemoglobin levels and can eliminate transfusion dependency in some cases of MDS for a period of time. ESAs of interest include, but are not limited to, erythropoietin-alpha, erythropoietin-beta, and darbepoetin.

[0074] In some embodiments, the subject is identified as a human patient with trisomy 8. In certain examples, the subject is a human patient with mosaic trisomy 8. In other examples, the subject is a human patient with non-mosaic trisomy 8.

[0075] In certain embodiments of the subject methods, the subject is classified as an IPSS low-risk or intermediate-1-risk MDS subject. Myelodysplastic syndrome (MDS) patients can be divided into lower-risk groups (IPSS low and intermediate-1 [INT-1]), in which apoptotic events in the bone marrow are widespread and the patient responds poorly to cytokines (including erythropoietin), and higher-risk groups (IPSS intermediate-2 [INT-2] and high), in which a block in the maturation of myeloid progenitor cells is the primary alteration. In some cases, transfusion dependence is a negative prognostic variable. Thus, in certain embodiments of the method, the subject is RBC transfusion-dependent. In some cases, transfusion-dependent subjects require RBC transfusions of about 4 units or more over an 8-week period, or 4 to 14 units over an 8-week period, or about 6 units or more per 8 weeks, prior to administration according to the subject methods. One unit of packed red blood cells (PRBCs) can be approximately 300 mL / unit. One unit of whole blood can be about 450-500 mL / unit.

[0076] The International Prognostic Scoring System (IPSS) is a system developed to stage MDS. The IPSS evaluates three factors: the percentage of leukemic blasts in bone marrow cells (scored on a scale of 0 to 2), chromosomal abnormalities (if any) in bone marrow cells (scored on a scale of 0 to 1), and the presence of one or more low blood counts (scored as 0 or 0.5). Each factor is given a score, with the lowest score having the best prognosis. The factor scores are then summed to obtain the IPSS score. The IPSS divides people with MDS into four groups: low risk, intermediate 1 risk, intermediate 2 risk, and high risk.

[0077] E. Telomerase inhibitors Any convenient telomerase inhibitor can find use in the subject method. In some embodiments, the telomerase inhibitor is an oligonucleotide having telomerase inhibitory activity, particularly the oligonucleotide defined in WO2005 / 023994 and / or WO2014 / 088785, the disclosures of which are incorporated herein by reference in their entirety. In some cases, one or more telomerase inhibitors (e.g., two or three telomerase inhibitors) can be administered to a mammal to treat hematological malignancies.

[0078] Imetelstat In certain embodiments, the telomerase inhibitor is imetelstat, including its tautomers and salts thereof, e.g., pharmaceutically acceptable salts. Imetelstat is a novel, first-in-class telomerase inhibitor with clinical activity in hematological malignancies (Baerlocher et al., NEJM 2015;373:920-928, Tefferi et al., NEJM 2015;373:908-919) (shown below). [ka] Here, "nps" represents a thiophosphoramidate bond, --NH--P(.dbd.O)(SH)--O--, which links the 3'-carbon of one nucleoside to the 5'-carbon of an adjacent nucleoside.

[0079] In certain examples, the telomerase inhibitor is imetelstat sodium, including its tautomers. Imetelstat sodium is the sodium salt of imetelstat, a synthetic lipid-conjugated 13-mer oligonucleotide N3'→P5'-thiophosphoramidate. Imetelstat sodium is a telomerase inhibitor that is a covalently lipidated 13-mer oligonucleotide (shown below) complementary to the human telomerase RNA (hTR) template region. The chemical name of imetelstat sodium is DNA, d(3'-amino-3'-deoxy-P-thio)(TAGGGTTAGACAA), 5'-[O-[2-hydroxy-3-(hexadecanoylamino)propyl]phosphorothioate], sodium salt (1:13) (SEQ ID NO: 1). Imetelstat sodium does not function through an antisense mechanism and therefore does not suffer from the side effects commonly observed with such therapies. [ka]

[0080] Unless otherwise indicated or clear from the context, a reference herein to imetelstat also includes its tautomers and salts, e.g., pharmaceutically acceptable salts. As stated, imetelstat sodium refers to the sodium salt of imetelstat. Unless otherwise indicated or clear from the context, a reference herein to imetelstat or imetelstat sodium also includes all of its tautomers.

[0081] Imetelstat and imetelstat sodium can be manufactured, formulated, or obtained as described elsewhere (see, for example, Asai et al., Cancer Res., 63:3931-3939 (2003), Herbert et al., Oncogene, 24:5262-5268 (2005), and Gryaznov, Chem. Biodivers., 7:477-493 (2010)). Unless otherwise indicated or clear from the context, a reference to imetelstat herein also includes its salts. As stated, imetelstat sodium refers to the sodium salt of imetelstat.

[0082] Imetelstat targets the telomerase RNA template and inhibits telomerase activity and cell proliferation in various cancer cell lines and tumor xenografts in mice. Phase 1 studies in patients with breast cancer, non-small cell lung cancer, and other solid tumors, multiple myeloma, or chronic lymphocytic leukemia have provided information on the drug's pharmacokinetics and pharmacodynamics. A subsequent Phase 2 study in patients with essential thrombocythemia demonstrated platelet-lowering activity associated with significant reductions in JAK2 V617F and CALR mutant allele burden. Imetelstat sodium is routinely administered intravenously. It is contemplated that other administration routes, such as intrathecal administration, intratumoral injection, subcutaneous administration, and oral administration, can also be used in the practice of the subject method. Imetelstat sodium can be administered at doses equivalent to those routinely used in clinical practice. In certain embodiments, imetelstat sodium is administered as described elsewhere herein.

[0083] F. Pharmaceutical Compositions For ease of administration, telomerase inhibitors (e.g., those described herein) can be formulated into various pharmaceutical forms for administration purposes. In some cases, the telomerase inhibitor is administered as a pharmaceutical composition. The carrier or diluent of a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient. The pharmaceutical composition may be in a unitary dosage form suitable for administration, particularly oral, rectal, transdermal, parenteral injection, or inhalation. In some cases, administration can be via intravenous injection. For example, when preparing a composition in oral dosage form, any of the usual pharmaceutical media, such as water, glycols, oils, alcohols, etc., may be employed for oral liquid preparations such as suspensions, syrups, elixirs, emulsions, and solutions; or solid carriers, such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrants, etc., may be employed for powders, pills, capsules, and tablets. Tablets and capsules are the most advantageous oral dosage unit forms due to their ease of administration; in these cases, solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier typically comprises, at least in large part, sterile water, although other ingredients, for example, to aid solubility, may be included. For example, injectable solutions may be prepared in which the carrier comprises saline, glucose solution, or a mixture of saline and glucose solution. For example, injectable solutions may be prepared in which the carrier comprises saline, glucose solution, or a mixture of saline and glucose solution. Injectable solutions containing the telomerase inhibitors described herein may be formulated in oil for long-acting action. Suitable oils for this purpose include, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long-chain fatty acids, and mixtures thereof with other oils. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, etc. may be employed. Also included are solid form preparations intended to be converted to liquid form preparations immediately prior to use.In compositions suitable for transdermal administration, the carrier optionally contains a penetration enhancer and / or a suitable wetting agent, optionally combined in minor proportions with suitable additives of any nature, which do not have a significant adverse effect on the skin. Such additives may facilitate application to the skin and / or may be useful in formulating the desired composition. The composition may be administered in various ways, for example, as a transdermal patch, as a spot-on, or as an ointment.

[0084] It is particularly advantageous to prepare the above-mentioned pharmaceutical composition into unit dosage form for the ease of administration and uniformity of dosage.Unit dosage form as used herein refers to a physically separate unit suitable as a single dosage form, and each unit contains a predetermined amount of active ingredient calculated to produce desired therapeutic effect in association with necessary pharmaceutical carrier.The example of this unit dosage form is tablet (including divided tablet or coated tablet), capsule, pill, powder packet, wafer, suppository, injection solution or suspension, etc., subcutaneous preparation, and their separate multiple doses.

[0085] To enhance the solubility and / or stability of the drugs described herein in the pharmaceutical composition, it may be advantageous to employ α-, β-, or γ-cyclodextrin or their derivatives, particularly hydroxyalkyl-substituted cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Cosolvents such as alcohols may also improve the solubility and / or stability of the telomerase inhibitor in the pharmaceutical composition.

[0086] Depending on the mode of administration, the pharmaceutical composition comprises 0.05 to 99% by weight, for example, 0.1 to 70% by weight, for example, 0.1 to 50% by weight, of a telomerase inhibitor described herein and 1 to 99.95% by weight, for example, 30 to 99.9% by weight, for example, 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

[0087] G. Administration and Dosing Regimen The administration frequency can be any frequency that reduces the severity of MDS symptoms (e.g., as described herein) without causing significant toxicity to the subject. For example, the administration frequency can be about once every two months to about once per week, alternatively about once per month to about twice per month, alternatively about once per six weeks, about once per five weeks, alternatively about once per four weeks, alternatively about once per three weeks, alternatively about once per two weeks, or alternatively about once per week. The administration frequency can remain constant or can vary over the course of treatment. A course of treatment with a composition comprising one or more telomerase inhibitors can include a rest period. For example, a composition comprising a telomerase inhibitor can be administered weekly for three weeks, followed by a two-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can affect the actual administration frequency used for a particular application. For example, the effective amount, the duration of treatment, the use of multiple therapeutic agents, the route of administration, and the severity of the MDS and related symptoms may require increased or decreased frequency of administration.

[0088] The effective period for administering a composition containing a telomerase inhibitor (e.g., imetelstat or imetelstat sodium) can be any period that reduces the severity of MDS symptoms (e.g., as described herein) without causing significant toxicity to the subject. Thus, the effective period can vary from one month to several months or years (e.g., one month to two years, one month to one year, three months to two years, three months to ten months, or three months to eighteen months). Generally, the effective period for treating MDS can range from two months to twenty months. In some cases, the effective period can last as long as the individual subject lives. Several factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of MDS and related symptoms.

[0089] In certain examples, can monitor the progress of treatment and the severity of one or more symptoms associated with MDS.Can use any method to determine whether the severity of MDS symptoms is reduced.For example, (for example, as described herein) can use biopsy technology to evaluate the severity of MDS symptoms.

[0090] The telomerase inhibitors used in the subject methods can be administered at any therapeutically effective dose, including doses corresponding to those routinely used in clinical practice. Specific dosage regimens (e.g., recommended effective doses) for known and approved anticancer drugs are known to physicians and are provided in product descriptions found, for example, in PHYSICIANS' DESK REFERENCE, 2003, 57th Ed., Medical Economics Company, Inc., Oradell, NJ, Goodman & Gilman's "THE PHARMACOLOGICAL BASIS OF THERAPEUTICS" 2001, 10th Edition, McGraw-Hill, New York, and / or are available from the Federal Drug Administration and / or are discussed in the medical literature.

[0091] In some embodiments, the dose of the telomerase inhibitor imetelstat sodium administered to a subject is about 1.0 mg / kg to about 13.0 mg / kg. In other embodiments, the dose of the telomerase inhibitor is about 4.5 mg / kg to about 11.7 mg / kg, or about 6.0 mg / kg to about 11.7 mg / kg, or about 6.5 mg / kg to about 11.7 mg / kg. In some embodiments, the dose of the telomerase inhibitor is at least about 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 ... mg / kg, 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, 11.2mg / kg, 11. Contains any of the following: 3mg / kg, 11.4mg / kg, 11.5mg / kg, 11.6mg / kg, 11.7mg / kg, 11.8mg / kg, 11.9mg / kg, 12mg / kg, 12.1mg / kg, 12.2mg / kg, 12.3mg / kg, 12.4mg / kg, 12.5mg / kg, 12.6mg / kg, 12.7mg / kg, 12.8mg / kg, 12.9mg / kg, or 13mg / kg.

[0092] In some embodiments, the effective amount of the telomerase inhibitor administered to an individual comprises at least about any of 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 4.7 mg / kg, 5 mg / kg, 6.0 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 some embodiments, the effective amount of the telomerase inhibitor administered to an individual is at least about any of 1 mg / kg, 2.5 mg / kg, 3.5 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 an individual comprises less than about any of 350 mg / kg, 300 mg / kg, 250 mg / kg, 200 mg / kg, 150 mg / kg, 100 mg / kg, 50 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 7.5 mg / kg, 6.5 mg / kg, 5 mg / kg, 3.5 mg / kg, 2.5 mg / kg, 1 mg / kg, or 0.5 mg / kg of telomerase inhibitor.

[0093] Exemplary dosing frequencies for pharmaceutical compositions containing a telomerase inhibitor include, but are not limited to, daily, every other day, twice a week, three times a week, weekly without a break, weekly for three weeks out of four, once every three weeks, once every two weeks, or weekly for two weeks out of three weeks. In some embodiments, the pharmaceutical composition is administered approximately once every week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. In some embodiments, the composition is administered at least about once, twice, three times, four times, five times, six times, or seven times a week (i.e., daily), or three times daily or twice daily. In some embodiments, the interval between each administration is less than 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 interval between each administration is greater than about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there are no breaks in the dosing schedule. In some embodiments, the interval between each administration is less than about 1 week.

[0094] A telomerase inhibitor such as imetelstat (e.g., imetelstat sodium) can be administered using any suitable method. For example, a telomerase inhibitor such as imetelstat (e.g., imetelstat sodium) can be administered intravenously over a period of time (e.g., 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours) once every four weeks. In some embodiments, imetelstat is administered intravenously at 7-10 mg / kg over approximately two hours once per week. In certain embodiments, imetelstat is administered intravenously at 2.5-7 mg / kg over approximately two hours once per three weeks. In embodiments, imetelstat is administered intravenously at 0.5-5 mg / kg over approximately two hours once per four weeks. In embodiments, imetelstat is administered intravenously at approximately 2.5-10 mg / kg over approximately two hours once per three weeks. Alternatively, imetelstat is administered intravenously at about 0.5 to 9.4 mg / kg over about two hours once every four weeks.

[0095] In certain embodiments of the method, imetelstat is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising intravenously administering about 7-10 mg / kg of imetelstat once every 4 weeks, about 7-10 mg / kg of imetelstat once every week for 4 weeks, about 2.5-10 mg / kg of imetelstat once every 3 weeks, or about 0.5-9.4 mg / kg of imetelstat once every 4 weeks. In certain examples, each dosing cycle comprises intravenously administering about 7-10 mg / kg of imetelstat once every 4 weeks. In some instances, each dosing cycle comprises intravenously administering about 7.5 mg / kg of imetelstat about once every 4 weeks.

[0096] In one embodiment of the invention, imetelstat is administered intravenously at a dosage of about 7-10 mg / kg of imetelstat once every four weeks after prior medication with an antihistamine, a corticosteroid, or both. In another embodiment, imetelstat is administered intravenously at a dosage of about 7.5 mg / kg, alternatively about 7.0 mg / kg to about 7.7 mg / kg of imetelstat once every four weeks after prior medication with an antihistamine, a corticosteroid, or both.

[0097] In certain embodiments, imetelstat is administered at a dosage of about 7.5 mg / kg, alternatively about 7.0 mg / kg to about 7.7 mg / kg, once every four weeks for at least three cycles, with the dosage then increased. In certain embodiments, the dosage of imetelstat is such that the ANC and platelet nadir are each about 1.5 x 10 9 / L~Approx. 75×10 9 The dose may be increased to about 9.4 mg / kg, alternatively to about 8.8 mg / kg to about 9.6 mg / kg, provided that the dose is not reduced by 100 mg / L and there is no Grade 3 or higher non-hematologic toxicity.

[0098] In one embodiment of the present invention, imetelstat sodium is administered intravenously once every four weeks at a dosage of about 4.5 to 11.7 mg / kg, e.g., about 7 to 10 mg / kg of imetelstat sodium. In another embodiment, imetelstat sodium is administered intravenously once every four weeks at a dosage of about 7.5 mg / kg, alternatively about 7.0 mg / kg to about 7.7 mg / kg.

[0099] It will be understood that cancer treatment sometimes involves multiple "rounds" or "cycles" of drug administration, with each cycle comprising one or more drug administrations according to a designated schedule (e.g., three consecutive days every three weeks; once a week, etc.). For example, anticancer drugs can be administered for one to eight cycles, or for a longer period. When a subject is administered two or more drugs (e.g., two drugs), each can be administered according to its own schedule (e.g., weekly, once every three weeks, etc.). It will be apparent that drug administration, even for drugs administered at different cycles, can be coordinated so that both drugs are administered at least some time on the same day, or alternatively, so that the drugs are administered at least some time on consecutive days.

[0100] As is understood in the art, if toxicity is observed or for the convenience of the patient, treatment with a cancer therapeutic agent can be temporarily interrupted and then resumed without departing from the scope of the present invention.

[0101] In certain embodiments, the present invention relates to a telomerase inhibitor for use in a method for treating MDS, the method comprising administering an effective amount of the telomerase inhibitor to a subject in need thereof, wherein the subject has not been treated with an agent selected from an HMA and lenalidomide. In other embodiments, the present invention relates to a telomerase inhibitor for use in a method for treating MDS, the method comprising administering an effective amount of the telomerase inhibitor to a subject in need thereof, wherein the subject has not been treated with an agent selected from an HMA, lenalidomide, and combinations thereof.

[0102] In certain embodiments, the present invention relates to a telomerase inhibitor for use in the method defined in any of the other embodiments.

[0103] H. Exemplary Embodiments Exemplary embodiments of the methods of treating MDS of the present invention are shown in Table 1 below, and include administering an effective amount of a telomerase inhibitor to a subject in need thereof, wherein the subject is not currently receiving treatment with an agent selected from a methylation inhibitor (HMA) and lenalidomide.

[0104] Exemplary embodiments include using any of the telomerase inhibitors in Table 1 to treat any one of the types of MDS shown in Table 1 in any one of the subjects shown in Table 1, where the subject has not received any one of the treatments shown in Table 1. In certain embodiments, one of the dosing regimens set forth in Table 1 is used. In other embodiments, the method may be used to treat any one of the types of MDS shown in Table 1 using imetelstat (imetelstat sodium) in any one of the subjects shown in Table 1, where the subject has not received any one of the treatments shown in Table 1. When imetelstat, e.g., imetelstat sodium, is used, any of the dosing regimens shown in Table 1 may be used. [Table 1-1] [Table 1-2]

[0105] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0106] Example 1: Efficacy and Safety in Transfusion-Dependent (TD) Patients with International Prognostic Scoring System (IPSS) Low-Risk / Intermediate-1-Risk Myelodysplastic Syndrome Who Are Relapsed or Refractory to Erythropoiesis-Stimulating Agent (ESA) Treatment Introduction An international clinical trial of imetelstat sodium in patients with RBC TD, ESA relapsed or refractory, or EPO (erythropoietin) >500mU / ml, and IPSS low-risk or intermediate-1 risk MDS. This example provides safety and efficacy findings for 177 patients. Results suggest improved efficacy of imetelstat among these patients.

[0107] method Study Design and Monitoring A randomized, double-blind, placebo-controlled trial was conducted at 77 sites in 17 countries. The trial was conducted in accordance with institutional and CONSORT (Consolidated Standards of Reporting Trials) guidelines and competent institutional laws. The institutional review board or ethics committee at each site approved the protocol. All patients provided written informed consent. The trial was designed in collaboration with an external steering committee, and an independent data safety monitoring committee monitored the trial. The sponsor provided guidance in the study design development and data collection and evaluation.

[0108] patient Eligible patients were aged 18 years or older, had MDS confirmed by BM aspirate and biopsy within 12 weeks prior to randomization by an independent central laboratory, had disease classified as low-risk or intermediate-1 risk by IPSS, had RBC-TD requiring ≥4 units for 8 of the 16 weeks prior to randomization, had MDS that was relapsed, refractory, or ineligible for ESAs (based on endogenous erythropoietin levels >500 mU / mL), had not received lenalidomide or hypomethylating agents, and were non-del(5q).

[0109] Test Design Patients were randomly assigned (based on a computer-generated schedule) in a 2:1 ratio to receive imetelstat or placebo, administered intravenously every 4 weeks until treatment discontinuation. Crossover was not permitted. Randomization was stratified by previous RBC transfusion burden (≥4 units to ≤6 units or >6 units for 8 of the 16 weeks prior to randomization) and IPSS risk group (low risk or intermediate risk). The starting dose of imetelstat was 7.5 mg / kg every 4 weeks. If patients developed grade 3 or 4 hematologic and nonhematologic toxicities, the dose was withheld and reduced as needed (Tables 2–4). [Table 2] IV indicates intravenous. [Table 3] [Table 4] Transfusion status was assessed at every scheduled and unscheduled visit. Disease assessments were performed every 12 weeks after the first dose until week 72, and then every 24 weeks until treatment discontinuation, and for discontinued patients, within 30 days of the last dose whenever possible. Treatment was discontinued for patients who experienced unacceptable toxicity, disease progression, withdrew consent, or met any discontinuation criteria. After treatment discontinuation, patients were evaluated every 12–16 weeks and followed up every 4–6 weeks for transfusions and disease status. (The study design is shown in Figure 1.)

[0110] Endpoint Measurement The primary endpoint was the rate of RBC-TI for at least 8 consecutive weeks. Key secondary endpoints included the 24-week TI rate, duration of TI, and hematologic response rate (hematologic improvement-red blood cells [HI-E] based on the modified International Working Group [IWG] 2006 criteria and HI-E according to the revised IWG 2018). Additional secondary endpoints included the relative change in the number of RBC transfusions patients received, the relative change in hemoglobin, and safety. Changes in mutation status and patient-reported fatigue (assessed by the Functional Assessment of Chronic Illness Therapy [FACIT] fatigue scale) over time were exploratory endpoints. Subgroup analyses were conducted, including the presence or absence of baseline RS, previous RBC transfusion burden (≥4 RBC units to ≤6 RBC units or >6 RBC units in 8 of the 16 weeks prior to randomization), and IPSS category low or intermediate risk. Subgroup analyses were also performed, including baseline mutation status, as well as telomerase activity (TA), telomere length (TL), and human telomerase reverse transcriptase (hTERT) levels. Peripheral hematology, bone marrow evaluation, mutation evaluation, and cytogenetic evaluation were performed centrally, and an independent review committee (IRC) assessed response. The 1-year RBC-TI rate was reported as a post-hoc analysis.

[0111] statistical analysis The data cutoff date for this primary analysis was 1 year after the last patient was randomized. All efficacy analyses of the primary and key secondary endpoints were conducted in the intention-to-treat population. The underlying hypothesis of this study was that imetelstat significantly improves the RBC-TI rate compared with placebo in patients with RBC-TD low-risk MDS. The percentages of patients with responses in the two treatment groups were compared using a stratified Cochran-Mantel-Haenszel test at a two-sided significance level of 0.05, adjusting for stratification factors, previous RBC transfusion burden, and IPSS low-risk or intermediate-1 risk category. Multiplicity was accounted for by a sequential gatekeeping approach for the primary and key secondary endpoints, which were tested sequentially in a prespecified order: 8-week TI followed by 24-week TI. The duration of RBC-TI was calculated using the Kaplan-Meier method. Safety analyses were conducted in the safety population (all patients who received at least one dose of study drug).

[0112] The study design is summarized in the following paragraphs.

[0113] Eligibility: The study eligibility requirements were as follows: IPSS low-risk or intermediate-1 risk MDS Relapsed or refractory to ESA or EPO >500mU / ml*. (*Received at least 8 weeks of ESA therapy (≥40,000U epoetin alfa, ≥30,000U epoetin beta, or 150mcg or equivalent darbepoetin alfa per week) without a ≥1.5g / dL increase in Hgb from ≥8 weeks of ESA therapy, or a ≥4 unit / 8 week reduction in RBC transfusion requirement or transfusion dependency, or a 1.5g / dL reduction in Hgb after hematologic improvement.) Transfusion dependent: ≥4 units of RBC for 8 weeks in the 16 weeks prior to the study Non-deleted 5q No prior treatment with lenalidomide or HMA.

[0114] One hundred seventy-eight patients were randomized in a 2:1 ratio to an imetelstat group or a placebo group: 118 patients received imetelstat and 59 patients received placebo (Figure 14). Details of patient disposition at the time of data cutoff are also shown in Figure 14, including 27 patients (23%) in the imetelstat group and 14 patients (24%) in the placebo group who were still receiving treatment at the time of data cutoff.

[0115] Stratification: Transfusion burden (4-6 units vs. >6 units) IPSS risk category (low risk vs. moderate risk)

[0116] Treatment: Patients in the imetelstat treatment group received imetelstat sodium at 7.5 mg / kg administered as a 2-hour IV infusion every 4 weeks. Supportive care, including RBC and platelet transfusions, myeloid growth factors (e.g., G-CSF (granulocyte colony-stimulating factor)), and iron chelation therapy, was administered as needed throughout the study at the investigator's discretion. The control group received corresponding treatment without imetelstat.

[0117] Endpoints and Analysis: Primary endpoint: 8-week RBC TI. The proportion of patients who were free of any RBC transfusion for at least 8 consecutive weeks from the start of the study (8-week TI). Key secondary endpoints: 24-week RBC TI: The proportion of patients who were free of any RBC transfusion for at least 24 consecutive weeks from the start of the study (24-week TI). ○ Duration of TI, ○ Hematological improvement - red blood cells (HI-E).

[0118] result Key Test Indicators The balanced study and treatment durations between the two arms are shown in Table 5 below. [Table 5] 8-week TI = Percentage of patients who have not received any RBC transfusion for at least 8 consecutive weeks from the start of the study

[0119] Baseline demographic and disease characteristics were comparable between arms, as shown in Table 6A below. Additional baseline characteristics are shown in Table 6B below. [Table 6A-1] [Table 6A-2] [Table 6B]

[0120] Overall, 178 patients were enrolled, with 118 randomly assigned to receive imetelstat and 60 to receive placebo (Figure 14). After a median follow-up of 18 months (range, 0.7-36.2), patients received a median of 8 treatment cycles of imetelstat (range, 1-34) and 8 treatment cycles of placebo (range, 1-30). The median treatment duration was 33.9 weeks (range, 0.1-141.1) for imetelstat and 28.3 weeks (range, 0.1-116) for placebo. The mean relative dose intensity to the starting dose of 7.5 mg / kg / cycle was 90% (range, 64-103) in the imetelstat group and 98% (range, 72-105) in the placebo group. Details of patient disposition at the time of data cutoff are shown in Figure 14 and include 27 patients (23%) in the imetelstat group and 14 patients (24%) in the placebo group who were still receiving treatment at the time of data cutoff.

[0121] Patient baseline characteristics were well balanced between groups (Table 6A). Additional baseline characteristics are shown in Table 6B. Overall, the median patient age was 72 years (range, 39–87 years), and 62% of patients were male. The median pretreatment hemoglobin was 7.9 g / dL (range, 5.3–10.1), and the median baseline serum erythropoietin was 184.1 mU / mL (range, 6–5514.0). Baseline IPSS risk categories were low risk for 67% of patients, intermediate risk for 33%, and MDS-RS for 62% of patients. Patients were heavily transfused at baseline, with a median previous RBC transfusion burden of 6.0 units / 8 weeks (range, 4–33). 47% of patients had a previous transfusion burden of >6 units / 8 weeks. Overall, 90% of patients had received a previous ESA and 6% had received previous luspatercept.

[0122] Patient disposition after a median follow-up of 18 months is shown in Table 7 below. [Table 7]

[0123] Primary endpoint: RBC-TI for ≥8 consecutive weeks Forty-seven patients (39.8%) in the imetelstat group achieved RBC-TI for at least 8 weeks compared with 9 patients (15.0%) in the placebo group (P<0.001, Figure 1A). With imetelstat treatment, RBC-TI was uninterrupted and sustained. 83% of 8-week RBC-TI responders had a single uninterrupted RBC-TI period (Figure 15). The primary endpoint was achieved in 45% (33 of 73) and 32% (14 of 44) of patients in the imetelstat group with and without RS, respectively, compared with 19% (7 of 37; P=0.016) and 9% (2 of 23; P=0.038) of patients in the placebo group (Figure 7A). Assessed based on prior RBC transfusion burden, RBC-TI ≥8 weeks occurred more frequently in the imetelstat group, in 45% (28 of 62) of patients with ≥4 units but ≤6 units / 8 weeks, and in 34% (19 of 56) of patients with >6 units / 8 weeks, compared with the corresponding frequency of 21% (7 of 33, P = 0.027) and 7% (2 of 27, P = 0.023) of patients in the placebo group. A significantly higher proportion of patients with IPSS low risk (40% vs. 20.5%, P = 0.034) and intermediate risk (40% vs. 5%, P = 0.004) had 8-week RBC-TI in the imetelstat group compared with the placebo group (Figure 7A).

[0124] Imetelstat treatment achieved the primary endpoint (8-week TI) with a highly statistically significant and clinically meaningful improvement in 8-week TI. The results are shown in Table 8 below. [Table 8]

[0125] Uninterrupted and sustained TI was achieved with imetelstat treatment, with 83% of 8-week TI responders having one uninterrupted TI period, as shown in FIG. 2.

[0126] The median duration of TI with imetelstat, as shown in Figure 3A, demonstrated highly statistically significant and clinically meaningful durability of TI during imetelstat treatment.

[0127] Imetelstat treatment met key secondary endpoints, i.e., highly statistically significant and clinically meaningful improvements in 24-week TI were observed, as shown in Table 9 below. [Table 9]

[0128] Key secondary and exploratory endpoints Uninterrupted RBC-TI for at least 24 weeks was demonstrated in 33 patients (28%) in the imetelstat group and 2 patients (3%) in the placebo group (P<0.001, Figure 4A). Among patients with RS, the 24-week RBC-TI rate was 33% in the imetelstat group compared with 5% in the placebo group, with corresponding values ​​of 20% and 0%, respectively, in patients without RS. Figure 7B shows a subgroup analysis of 24-week RBC-TI.

[0129] The rates of 1-year or longer TI in imetelstat-treated patients compared with placebo are shown in Figure 4B, and the percentages of patients exhibiting 8-week or longer TI and 24-week or longer TI who also exhibited 1-year or longer TI are shown in Figure 4C. Notably, 21 of 118 patients (17.8%) who received imetelstat achieved 1-year or longer TI (95% CI, 11.4-25.9), while 1 of 60 patients (1.7%; 95% CI, 0-8.9) who received placebo and supportive care achieved 1-year or longer TI. Furthermore, 1-year or longer TI was achieved by 44.7% and 63.6% of 8-week or longer and 24-week or longer TI, respectively, of imetelstat-treated responders.

[0130] The correlation between baseline TA, TL, and hTERT levels and clinical benefit demonstrated that imetelstat had a higher RBC-TI rate than placebo, regardless of baseline TA, TL, and hTERT expression. However, patients with higher TA, hTERT, or shorter TL were more sensitive to imetelstat treatment, showing more pronounced differences in RBC-TI over 8 and 24 weeks when treated with imetelstat versus placebo than patients with lower TA and hTERT, or longer TL (Figures 7C-7D). These results are consistent with telomere biology in cancer cells and provide evidence of imetelstat's precise MOA via telomerase inhibition. This result also supports findings from a phase 2 study of imetelstat in patients with myelofibrosis.

[0131] At further follow-up, the 1-year RBC-TI rate was 17.8% (21 / 118) of patients in the imetelstat group compared with 1.7% (1 / 60) of patients in the placebo group ( P = 0.002, Figure 4 ).

[0132] In patients meeting the primary endpoint, the median duration of RBC-TI in the imetelstat group was 51.6 weeks (95% CI, 26.9 to 83.9) compared with 13.3 weeks (95% CI, 8.0 to 24.9) in the placebo group, a difference of 38.3 weeks with a hazard ratio (HR) = 0.23 (95% CI, 0.091 to 0.571; P < 0.001; Fig. 3 ).

[0133] The HI-E rates according to the IWG 2006 and IWG 2018 criteria are shown in Table 10. More patients achieved an increase in hemoglobin of 1.5 g / dL or greater over 8 weeks with imetelstat [40, 34% (95% CI, 25 to 43)] compared with placebo [6, 10% (95% CI, 3.8 to 20.5); P < 0.001]. HI-E according to the IWG 2018 criteria was experienced by 50 patients (42%) in the imetelstat group compared with 8 patients (13%) in the placebo group (P < 0.001). [Table 10]

[0134] Among patients who achieved an 8-week TI, the median increase in blood hemoglobin from pretreatment to peak during the longest response period was 3.55 g / dL in the imetelstat group compared with 0.80 g / dL in the placebo group (Figure 4A). Over time, patients in the imetelstat group experienced a greater increase in mean hemoglobin levels than patients in the placebo group (P<0.001, Figure 4A). During the study, patients in the imetelstat group had a significant reduction in RBC transfusion units over time compared with patients in the placebo group (P=0.042, Figure 3).

[0135] Of the randomized patients, 22% in each group had cytogenetic abnormalities at baseline. Complete or partial cytogenetic responses were observed by an independent review committee in nine patients (35%) in the imetelstat group and two patients (15%) in the placebo group (Table 11). Of the 21 1-year or longer TI responders treated with imetelstat, seven (33%) had cytogenetic abnormalities at baseline and one or more post-treatment cytogenetic evaluations. Of these, four (57%) had a cCR, ​​two (29%) had a cPR, and one patient (19%) did not meet response criteria. [Table 11]

[0136] Among patients with evaluable mutation data, reductions in VAF for SF3B1, TET2, DNMT3A, and ASXL1 genes, which are commonly mutated in MDS, were greater with imetelstat than with placebo (Figure 12A), and VAF reductions correlated with increased achievement of 8-week RBC-TI (Table 12), longer RBC-TI duration (Figure 12B), and increased mean hemoglobin (Figure 12C) in patients treated with imetelstat. Similar results showed reduced TET2 VAF and / or reduced DNMT3A VAF correlated with increased achievement of longer RBC-TI duration and increased mean hemoglobin in patients treated with imetelstat. (Figures 17A-17D.) [Table 12]

[0137] P values ​​were determined using the Cochran Mantel-Haenszel test stratified for previous RBC transfusion burden (≤6 units or >6 units RBCs / 8 weeks) and baseline IPSS risk score (low risk or intermediate risk). The median TI duration (Kaplan-Meier estimate of RBC TI duration) for imetelstat patients who achieved a 24-week TI was 80.0 weeks (51.6, NE). Approximately 70% of imetelstat-treated patients who achieved an 8-week TI also achieved a 24-week TI.

[0138] When TI rates were evaluated over time, an increased magnitude of benefit with longer TI was observed with imetelstat compared with placebo, as shown in Figure 4 .

[0139] As shown in Figure 5, a statistically highly significant increase in hemoglobin levels was observed in patients treated with imetelstat compared to placebo. For 8-week TI responders, the median hemoglobin increase with imetelstat was 3.6 g / dL compared to 0.8 g / dL with placebo. Also, for 8-week TI responders, the median peak hemoglobin was 11.3 g / dL compared to 8.9 g / dL with placebo. This analysis was performed during the TI period.

[0140] As shown in Figure 6, a statistically significant reduction in the number of RBC units transfused was observed in patients treated with imetelstat compared to placebo.

[0141] Additional secondary endpoints, specifically hematological improvement-red blood cells (HI-E), were analyzed. Using the updated IWG 2018 criteria, a highly statistically significant HI-E was observed. Although the HI-E using the protocol-specified IWG 2006 criteria was not statistically significant, the evolution of the newer HI-E criteria (IWG-2018) places more emphasis on durability by measuring responses beyond 16 weeks. [Table 13]

[0142] As shown in Figure 7A, statistically significant 8-week TI rates (p<0.05) and similar degrees of clinical benefit were demonstrated across all subgroups.

[0143] The RS+ and RS- groups show statistically significant improvements at both the 8-week and 24-week TIs, as shown in FIG.

[0144] As shown in Table 14, the primary endpoint was met. 39.8% vs. 15.0% of patients receiving imetelstat or placebo, respectively, achieved 8-week TI. p<0.001. The rate of 8-week TI was statistically significantly higher with imetelstat versus placebo, with 83% of 8-week RBC-TI responders having a single uninterrupted RBC-TI period (Figure 15). As shown in Table 14, the median TI duration was significantly longer with imetelstat versus placebo: 51.6 weeks vs. 13.3 weeks, p<0.001. Patients receiving imetelstat had significantly higher mean hemoglobin over time (p<0.001) and fewer transfusions (p=0.042) than placebo patients.

[0145] The primary endpoint was achieved in 45% (33 of 73) and 32% (14 of 44) of patients in the imetelstat group with and without RS, respectively, compared with 19% (7 of 37; P = 0.016) and 9% (2 of 23; P = 0.038) of patients in the placebo group (Figure 7A). When assessed based on prior RBC transfusion burden, RBC-TI at 8 weeks or longer occurred more frequently in the imetelstat group: 45% (28 of 62) of patients with ≥ 4 units to ≤ 6 units / 8 weeks, and 34% (19 of 56) of patients with > 6 units / 8 weeks, compared with the corresponding frequency of 21% (7 of 33; P = 0.027) and 7% (2 of 27; P = 0.023) of patients in the placebo group (Figure 7A). A significantly higher proportion of patients with IPSS low risk (40% vs. 20.5%, P = 0.034) and intermediate 1 risk (40% vs. 5%, P = 0.004) had 8-week RBC-TI in the imetelstat group compared with the placebo group (Figure 7A). [Table 14]

[0146] 9 shows that patients who responded to imetelstat therapy showed a 50% or greater reduction in VAF for the genes SF3B1, TET2, DNMT3A, and ASXL1. These data indicate that a 50% or greater reduction in VAF for one or more of the genes SF3B1, TET2, DNMT3A, and ASXL1 can be used to identify and specifically treat MDS patients, as such patients are likely to benefit from treatment with a telomerase inhibitor.

[0147] VAF reduction was significantly greater in patients treated with imetelstat than in those receiving placebo in three of four genes frequently mutated in MDS: SF3B1 (p<0.001), TET2 (p=0.032), DNMT3A (p=0.019), and ASXL1 (p=NS). SF3B1 VAF reduction correlated with longer TI duration (r=0.549, p<0.001).

[0148] No new safety signals were identified. The most common grade 3 / 4 adverse events were thrombocytopenia and neutropenia, and the rates of grade 3 or higher bleeding and infection during imetelstat and placebo treatment were similar. Cytopenias were manageable in imetelstat-treated patients; they were short-lived and resolved to grade 2 or lower within 4 weeks in more than 80% of patients.

[0149] Grade 3-4 thrombocytopenia and neutropenia occurred in 14 (67%) and 20 (95%) patients with TI >1 year. The mean (SD) duration of grade 3 / 4 neutropenic and thrombocytopenic events was 1.78 (1.58) weeks and 2.25 (2.48) weeks, respectively. 81% of grade 3 / 4 neutropenic events and 89% of grade 3 / 4 thrombocytopenic events resolved to grade 2 or less within 4 weeks.

[0150] These data also demonstrate that imetelstat treatment for MDS reduces the depth, breadth, and durability of TI. Highly statistically significant (p<0.001) and clinically meaningful improvements in 8-week, 16-week, and 24-week TI were observed in imetelstat-treated patients compared with placebo. The median TI duration in imetelstat-treated patients approached 1 year.

[0151] A highly statistically significant (p<0.001) and clinically meaningful increase in mean change in hemoglobin levels over time was observed for imetelstat-treated patients versus placebo.

[0152] HI-E according to the IWG 2018 criteria showed a highly statistically significant (p<0.001) and clinically meaningful improvement in imetelstat-treated patients compared with placebo.

[0153] Relative to placebo, statistically significant (p<0.05) and clinically meaningful 8-week TI rates were achieved for both imetelstat-treated patients with high and very high transfusion burden, IPSS low and intermediate 1 risk, and RS+ and RS-.

[0154] Clinical and molecular evidence supporting the MDS disease-modifying potential of imetelstat includes a median 1-year TI duration in imetelstat 8-week TI responders, a median increase in hemoglobin levels of 3.6 g / dL in imetelstat 8-week TI responders, and a 50% or greater reduction in VAF in SF3B1, TET2, DNMT3A, and ASXL1 mutations.

[0155] Thus, imetelstat treatment demonstrated clinically meaningful increases in 8- and 24-week TI rates and TI duration, increases in hemoglobin, and reductions in VAF in patients with ESA R / R non-del(5q) lower-risk myelodysplastic syndromes (LR-MDS) who were not receiving len / HMA and had severe RBC TD.

[0156] The primary therapeutic goal in patients with LR-MDS is to alter disease biology by eradicating the malignant clone without sacrificing toxicity. MDS-initiating cells, carrying cytogenetic abnormalities, mutant alleles, or both, arise from malignant stem and progenitor cells. SF3B1, involved in RNA splicing, and TET2, involved in DNA methylation, are recurrently mutated genes in LR-MDS, which can be quantified by measuring changes in VAF to indicate disease burden. In a phase 3 trial, treatment with imetelstat demonstrated statistically significant and clinically meaningful efficacy over placebo, with a 40% TI rate over 8 weeks.

[0157] As described above, patients with severe RBC-TD-ESA relapsed / refractory or ineligible non-del(5q)LR-MDS who had not received lenalidomide and a hypomethylating agent (len / HMA) received a 2-hour infusion of imetelstat 7.5 mg / kg or placebo every 4 weeks. An independent review committee assessed abnormal cytogenetic profiles in bone marrow samples obtained before treatment and every 24 weeks after treatment to evaluate cytogenetic response as complete or partial remission. Of the 178 randomized patients, 22.0% in the imetelstat group and 21.7% in the placebo group had baseline cytogenetic abnormalities and posttreatment samples. Cytogenetic responses were observed in 9 / 26 patients in the imetelstat group (34.6%, 95% CI=17.2–55.7) and 2 / 13 patients in the placebo group (15.4%, 95% CI=1.9–45.5).

[0158] In patients with ≥5% variant alleles at baseline and at least one post-baseline assessment, VAF changes were assessed from blood samples taken before treatment and every 12 weeks after treatment. Group comparisons were assessed using t-tests, associations were assessed using Fisher's exact test, and correlations were assessed using linear regression. Imetelstat-treated patients demonstrated a higher rate of ≥50% VAF reductions in SF3B1, TET2, DNMT3A, and ASXL1 mutations compared with placebo (Figure 9). Patients achieving ≥8-week, ≥24-week, and ≥1-year TI in the imetelstat group were enriched for patients with ≥50% reductions in SF3B1 and TET2 VAF compared with placebo (Figure 10). Additionally, both 8-week and 24-week TI responders in the imetelstat arm had significantly greater reductions in SF3B1 VAF or TET2 VAF compared with non-responders (p<0.001 in all cases). Furthermore, compared with subjects who did not achieve a 50% or greater reduction, patients with a 50% or greater VAF in SF3B1 had significantly higher rates of TI responders at 8 weeks (82.6% vs. 38.2%), 24 weeks (69.6% vs. 23.6%), and 1 year (47.8% vs. 5.5%) (p<0.001 in all cases). Importantly, greater reductions in SF3B1 VAF in the imetelstat arm correlated significantly with increases in hemoglobin; r=-0.626, p<0.001, and longer TI duration; r=-0.549, p<0.001 (Figures 12A-12C).

[0159] Thus, in a phase 3 imetelstat study, patients with more severely RBC TD, ESA-R / R / ineligible, non-del(5q) LR-MDS who were not receiving len / HMAs and were treated with imetelstat experienced cytogenetic responses and reductions in SF3B1, TET2, DNMT3A, and ASXL1 mutation burden compared with placebo. Furthermore, reductions in SF3B1 VAF correlated with clinically meaningful endpoints of increased hemoglobin and TI duration. Combined with robust rates of uninterrupted and durable TI, this may indicate improvement in the ineffective erythropoiesis characteristic of LR-MDS, suggesting the potential of imetelstat to modify disease biology in these patients.

[0160] As noted above, the phase 2 imetelstat study demonstrated that treatment with imetelstat resulted in prolonged and durable TI across a wide range of severely RBC TD ESA-relapsed / refractory non-del(5q)LR-MDS patients who were lenalidomide- and hypomethylating agents (len / HMA)-naive.

[0161] As mentioned above, the primary endpoint was the 8-week TI rate, and subgroup analyses included IPSS risk, previous transfusion burden, and RS status. Secondary endpoints included the 24-week TI rate, TI duration, and hematologic improvement-red blood cell (HI-E) rate. Change in VAF was exploratory. The primary and key secondary endpoints were compared using the Cochran-Mantel-Haenszel test stratified by previous transfusion burden and IPSS category. TI duration was calculated using the Kaplan-Meier method and compared using the stratified log-rank test.

[0162] Imetelstat treatment met the primary endpoint. 47 patients (39.8%) receiving imetelstat achieved 8-week TI compared with 9 (15.0%) receiving placebo; p<0.001. The 8-week TI rate was also significantly higher with imetelstat compared with placebo across subgroups, including RS-negative patients. The median TI duration (95% CI) was 51.6 (26.9-83.9) weeks for imetelstat and 13.3 (8.0-24.9) weeks for placebo; p<0.001. Thirty-three patients (28.0%) receiving imetelstat achieved 24-week TI compared with 2 (3.3%) receiving placebo; p<0.001. At an additional 3-month follow-up, 21 patients (17.8%) on imetelstat achieved a TI of 1 year or longer compared with 1 patient (1.7%) on placebo (p=0.002), representing 63.6% of imetelstat TI responders at 24 weeks or longer (Figure 11). The HI-E rate (2018 IWG, weighting responses >16 weeks) was 42.4% for imetelstat compared with 13.3% for placebo (p<0.001). Patients receiving imetelstat had significantly higher mean hemoglobin (p<0.001) and fewer transfusions (p=0.042) over time than placebo patients. VAF reductions in three genes frequently mutated in MDS were significantly greater in patients treated with imetelstat than in placebo: SF3B1 (p<0.001), TET2 (p=0.032), DNMT3A (p=0.019), and ASXL1 (p=NS). Reductions in SF3B1 VAF correlated with increased hemoglobin (p<0.001) and longer TI duration in imetelstat-treated patients. p<0.001.

[0163] Imetelstat demonstrated statistically significant and clinically meaningful efficacy, with robust 8-week, 24-week, and 1-year TI rates and extended uninterrupted TI duration. In this LR-MDS patient population, nearly one-fifth of patients treated with imetelstat achieved uninterrupted TI for 1 year or longer, representing substantial relief from transfusion-related complications. VAF reduction and its correlation with clinical endpoints, including durable TI, support the disease-modifying potential of imetelstat. Safety results are consistent with previous reports. Imetelstat treatment offers significant clinical benefit to a population of severely TD LR MDS patients in need of novel therapies.

[0164] safety The most frequently reported AEs were neutropenia and thrombocytopenia. AEs of any grade occurring in 10% or more of patients in either study group are shown in Table 15. Most non-hematologic adverse events (AEs) occurred at low grade. [Table 15]

[0165] Overall, 107 patients (91%) who received imetelstat and 28 patients (48%) who received placebo had grade 3 or 4 AEs (Table 16). A total of 38 patients (32%) who received imetelstat had at least one serious AE compared with 13 patients (22%) who received placebo (Table 16). [Table 16]

[0166] The most common grade 3-4 treatment-emergent hematologic AEs in the imetelstat group were neutropenia and thrombocytopenia in 80 (68%) and 73 (62%) patients, respectively. The median duration of neutropenia was 1.9 weeks, and 81% of grade 3-4 events resolved to grade 2 or less within 4 weeks. The median duration of thrombocytopenia was 1.4 weeks, and 86% of grade 3-4 events resolved to grade 2 or less within 4 weeks (Table 17). [Table 17]

[0167] The majority of patients who experienced these events did so within the first three treatment cycles (Table 18). [Table 18]

[0168] The majority of dose reductions and delays in the imetelstat group were due to neutropenia (33.1% and 50.8%, respectively) and / or thrombocytopenia (22.9% and 46.6%, respectively). Clinical outcomes from grade 3-4 hemorrhagic events, infections, and febrile neutropenic cytopenias were similar in patients treated with imetelstat and placebo (Table 19). [Table 19]

[0169] Table 20 below provides certain hepatic events observed in study subjects. [Table 20]

[0170] Fifty-eight patients (49%) receiving imetelstat and four patients (7%) receiving placebo had dose reductions (Table 21, Figure 16). [Table 21]

[0171] Overall, 19 patients (16%) receiving imetelstat discontinued treatment due to AEs, compared with none receiving placebo. AEs leading to treatment discontinuation included neutropenia (5% of patients) and thrombocytopenia (3% of patients; Table 22). [Table 22-1] [Table 22-2]

[0172] Seven patients (6%) in the imetelstat group and five patients (8%) in the placebo group discontinued treatment due to progressive disease: two patients (2%) in the imetelstat group and one patient (2%) in the placebo group, respectively, due to progression of acute myeloid leukemia. A total of 27 patients died during the study: 19 (16%) in the imetelstat group and eight (13%) in the placebo group, with only one death in each group occurring within the treatment period (one imetelstat patient due to neutropenic sepsis caused by the study drug and one placebo patient due to aortic stenosis, both unrelated to the study drug).

[0173] Example 2: Clinical Benefit of Imetelstat Treatment: Sustained and Significant Improvement in Fatigue Patients with LR-MDS and anemia experience severe fatigue that adversely affects overall function and daily life. Fatigue is also a commonly reported adverse event associated with treatments for LR-MDS aimed at minimizing transfusions and improving patient-reported outcomes (PROs). The Phase 3 study of imetelstat described in Example 1 assessed patient-reported fatigue (rate of worsening / improvement) during treatment with imetelstat or placebo.

[0174] To measure patient-reported fatigue, data were collected using the validated Functional Assessment of Chronic Illness Therapy (FACIT) Scale and analyzed for all randomized patients for whom fatigue data were available at baseline (PRO population). The FACIT is a 13-item questionnaire measuring fatigue during daily activities. The rate of sustained meaningful regression / improvement was defined as the percentage of patients with a 3-point decrease / increase in the FACIT fatigue scale (0-52) over two or more consecutive treatment cycles. Additionally, time to regression / improvement was estimated by Kaplan-Meier analysis. Sensitivity analyses were performed using alternative definitions of meaningful regression in the intention-to-treat population.

[0175] result: The PRO population included 118 imetelstat patients and 57 placebo patients. Completion rates for all PRO items exceeded 80% at most visits throughout the study in both groups. At baseline, the mean age was 71 years (range, 39-87), Eastern Cooperative Oncology Group performance status was 0 (36.0%), 1 (60.6%), or 2 (3.4%), and 62.3% of patients were male. The proportion of patients who experienced any episode of persistent and meaningful regression in fatigue during the study was 43.2% in the imetelstat group and 45.6% in the placebo group. Results of the sensitivity analysis were similar to those of the primary analysis. Overall, 50.0% of patients in the imetelstat group reported persistent and meaningful improvement in fatigue compared with 40.4% of patients in the placebo group. The median time to reporting the first sustained and meaningful improvement in fatigue was shorter with imetelstat than with placebo: 28.3 vs. 65.0 weeks, HR = 1.34 (95% CI, 0.82-2.20). After 12 weeks, more imetelstat patients than placebo reported improvement on the FACIT fatigue scale (Figure 13A). In addition, subsequent analysis showed that IWG 2006 criteria response TI and HI-E of 8 weeks or more and 24 weeks or more were highly significant and consistently correlated with sustained and meaningful improvements in fatigue scores in imetelstat-treated subjects, but not in the placebo group (Figure 13B).

[0176] Conclusion: In this severely transfusion-dependent (TD) population, both imetelstat- and placebo-treated patients reported similar fatigue regression rates, suggesting that imetelstat did not worsen the regression rates reported with currently available treatments. After 12 weeks, greater improvements in fatigue were reported with imetelstat compared with placebo. Imetelstat patients were more likely to have and experience sustained, meaningful improvements in fatigue more quickly than placebo patients. Finally, the clinical benefit of imetelstat treatment is demonstrated by the robust correlation between TI and HI-E responses and sustained, meaningful improvements in fatigue.

[0177] Example 3: Additional Information About the Phase 3 Clinical Trial The following table (Table 23) provides a list of the primary, secondary, and exploratory endpoints evaluated in the studies described in the Examples herein. [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4]

[0178] Additional information about this method Study inclusion criteria Males or females aged 18 years or older (or the legal age of consent in the jurisdiction where the study was conducted): In this phase 3 study, a diagnosis of MDS according to WHO criteria was confirmed by bone marrow aspirate and biopsy within 12 weeks before randomization. Baseline bone marrow aspirate and biopsy samples were submitted to an Independent Central Pathology Reviewer for diagnostic confirmation. A central laboratory review was required to confirm the diagnosis before randomization.

[0179] IPSS low-risk or intermediate-1 risk MDS: RBC transfusion dependence was defined as requiring a transfusion of at least 4 RBC units over an 8-week period during the 16 weeks prior to randomization, and pre-transfusion Hb must be ≤9.0 g / dL to count toward the total of 4 units.

[0180] Have MDS that has relapsed / refractory to ESA treatment, as defined by meeting any one of the following criteria: Received at least 8 weeks of treatment with a minimum weekly dose of epoetin alfa 40,000 U, epoetin beta 30,000 U, or darbepoetin alfa 150 mcg (or equivalent agent / dose) without an Hb increase of 1.5 g / dL or greater or a decrease in RBC transfusion requirements of at least 4 units over the 8 weeks

[0181] Unless otherwise stated, transfusion dependent or Hb reduction of 1.5 g / dL or greater after hematologic improvement from at least 8 weeks of treatment with both of the inclusion criteria discussed in the preceding paragraph.

[0182] Endogenous serum EPO level 500 mU / mL: Adequate iron stores defined as transferrin saturation >20% and serum ferritin >400 ng / mL measured within the screening period, or adequate iron stores demonstrated by a recent (within 12 weeks prior to randomization) bone marrow examination with iron staining.

[0183] ECOG performance status of 0, 1, or 2: Hematological laboratory values ​​within the following limits: Independent of growth factor support, 1.5 x 10 9 ANC of / L or more, Independently of platelet transfusion support, platelets 75 × 10 9 / L or more platelets.

[0184] Biochemical laboratory values ​​should be within the following limits: AST, ALT, and ALP less than or equal to 2.5 times the upper limit of normal (×ULN).

[0185] Serum creatinine below 2.0 × ULN.

[0186] Total bilirubin ≤ 3 x ULN and direct bilirubin ≤ 2 x ULN (unless due to Gilbert's syndrome, ineffective erythropoiesis due to MDS, or hemolysis due to RBC transfusion): Women of childbearing potential who practiced highly effective contraception in accordance with local regulations regarding the use of contraceptive methods for patients participating in clinical studies were eligible: established use of oral, injectable, or implanted hormonal contraceptive methods; placement of an intrauterine device or intrauterine system; barrier methods: condoms in conjunction with spermicidal foam / gel / film / cream / suppositories or occlusive caps (diaphragms or cervical / fornix caps) in conjunction with spermicidal foam / gel / film / cream / suppositories; sterilization of the male partner (a partner who has undergone a vasectomy must be the patient's only partner); and true abstinence (if this was consistent with the patient's preferred, usual lifestyle). For women, these restrictions applied for one month after the end of medication. If fertility changed after the start of the study (e.g., a woman who was not sexually active with the opposite sex became active, or a premenstrual woman experienced menarche), women must have initiated the above highly effective contraceptive methods.

[0187] Women of childbearing potential must have a negative serum (β-human chorionic gonadotropin [β-hCG]) or urine pregnancy test at screening and agree to be tested on day 1 of every cycle and at the end of the study (30 days after the last dose).

[0188] Men who were sexually active with women of childbearing potential and who did not have a vasectomy had to agree to use a barrier method of contraception, e.g., condoms in conjunction with spermicidal foam / gel / film / cream / suppositories, or occlusive caps (diaphragms or cervical / fornix caps) in conjunction with spermicidal foam / gel / film / cream / suppositories, and all men had to refrain from donating sperm during the study. For men, these restrictions applied for 3 months after the end of medication.

[0189] Each patient (or their legally acceptable representative) signed an informed consent form (ICF), indicating that they understood the purpose of the study and the procedures involved and were willing to participate in the study.

[0190] Study exclusion criteria Patients with known allergy, hypersensitivity, or intolerance to imetelstat or any excipients in imetelstat drug formulations.

[0191] Patients who received an investigational or investigational drug within 30 days prior to C1D1 (Part 1) or randomization (Part 2) (defined in Section 3.1), or who used an invasive investigational medical device, or who are currently enrolled in an investigational study.

[0192] Prior treatment with imetelstat: received more than 30 mg / day of prednisone or equivalent corticosteroids or growth factor treatment within 4 weeks prior to C1D1 (part 1) or randomization (part 2); prior treatment with a methylation inhibitor (e.g., azacitidine, decitabine); prior treatment with lenalidomide, thalidomide, or other thalidomide analogs; received ESA or any anti-MDS therapy, chemotherapy, immunomodulatory therapy, or immunosuppressive therapy within 4 weeks (8 weeks for long-acting ESAs) prior to C1D1 (part 1) or randomization (part 2); history of hematopoietic stem cell transplantation; anemia attributable to factors other than MDS (including hemolysis, chronic renal failure, hepatitis, and gastrointestinal bleeding); major surgery (excluding placement of vascular access and other minor surgical procedures) within 4 weeks prior to C1D1 (Part 1) or randomization (Part 2); Diagnosis or treatment of malignancies other than MDS, except for the following: Malignancies treated with curative intent and with no known active disease for ≥3 years prior to C1D1 (Part 1) or randomization (Part 2).

[0193] Adequately treated non-melanoma skin cancer or lentigo maligna with no evidence of disease.

[0194] Sufficiently treated in situ cervical cancer with no evidence of disease.

[0195] Clinically significant cardiovascular disease, such as uncontrolled or symptomatic arrhythmia, congestive heart failure, or myocardial infarction within 6 months of C1D1 (Part 1) or randomization (Part 2), or any class 3 (moderate) or class 4 (severe) heart disease as defined by the New York Heart Association Functional Classification.

[0196] Known history of human immunodeficiency virus (HIV) or any uncontrolled active systemic infection requiring IV antibiotics.

[0197] Active systemic hepatitis infection requiring treatment (carriers of hepatitis virus were allowed to participate in the study), or known acute or chronic liver disease, including cirrhosis.

[0198] Women who were pregnant, breastfeeding, or planning to become pregnant while enrolled in the study or within 1 month of stopping medication.

[0199] Patients were men planning to father a child while enrolled in the study or within 3 months of completing medication.

[0200] Any life-threatening disease, medical condition, or organ system dysfunction that, in the opinion of the investigator, could compromise patient safety, interfere with imetelstat metabolism, or place the study results at undue risk; the patient had any condition where, in the opinion of the investigator, participation may not be in the patient's best interest (e.g., compromise well-being) or may preclude, limit, or confound protocol-specified evaluations.

[0201] Patients have previously been assessed as having IPSS intermediate 2 or high-risk MDS.

[0202] Patients with del(5q) karyotype.

[0203] Patients with MDS / myeloproliferative neoplasm overlap syndrome.

[0204] HI-E according to IWG 2006 standard The proposed modified International Working Group response criteria for hematologic improvement are shown in Table 24. [Table 24]

[0205] HI-E according to IWG 2018 Criteria 2 The proposed modified International Working Group response criteria for response assessment are provided below in Table 25. [Table 25]

[0206] Defining the endpoint The 8-week RBC-TI rate was the proportion of patients who did not receive any RBC transfusions during any consecutive 8-week period (56 days) starting from Study Day 1 through subsequent anticancer therapy (if any). The start date of the 8-week period of transfusion independence was between randomization and 30 days after the last dose, the end-of-treatment visit, or 31 days after randomization if randomized but untreated. Study Day 1 was defined as the date of randomization for patients enrolled in the Phase 3 trial.

[0207] The key secondary endpoint, TI rate, was the proportion of patients who did not receive any RBC transfusions for any consecutive 16-week, 24-week, or 1-year period beginning on study day 1.

[0208] The key secondary endpoint of RBC-TI, duration, was from the first day of the longest RBC-TI period to the date of the first RBC transfusion after the start of the TI period.

[0209] Key secondary endpoint: Rate of hematologic improvement, including HI-E, according to modified IWG 2006.

[0210] Key secondary endpoints, complete remission (CR), partial remission (PR), or marrow complete remission (mCR): Proportion of patients achieving CR, PR, or mCR according to modified IWG 2006.

[0211] Exploratory endpoints: Mutation status at baseline and change over time assessed, including response (CR, PR, mCR, HI-E[Hb]) or suspected progressive disease.

[0212] Exploratory endpoint, patient-reported fatigue: The rate of sustained meaningful regression / improvement was defined as the percentage of patients who had a 3-point decrease / increase in the Functional Assessment of Chronic Illness Therapy (FACIT) fatigue scale (0-52) over two or more consecutive treatment cycles.

[0213] CR indicates complete response, Hb indicates hemoglobin, HI indicates hematological improvement, HI-E indicates hematological improvement-erythrocytes, IWG indicates International Working Group, PR indicates partial response, and RBC indicates red blood cells.

[0214] Additional statistical methods Changes over time in transfusion units and hemoglobin levels were compared by mixed models for repeated measures, with weeks since randomization, prior transfusion burden, lowest hemoglobin in the 8 weeks before first dose (for hemoglobin), and treatment arm as independent variables with an autoregressive moving-average covariance structure.

[0215] Meaningful Improvement on FACIT Fatigue: Supportive psychometric evidence for the use of FACIT Fatigue in the context of LR-MDS confirms the previously published threshold of a 3-point change for meaningful within-patient change on FACIT Fatigue.

[0216] FACIT stands for Functional Assessment of Chronic Illness Therapy, and LR-MDS stands for Reduced Risk of Myelodysplastic Syndrome.

[0217] Gene mutation analysis Blood samples were collected approximately every 12 weeks at baseline and during study visits. Following DNA extraction from white blood cells, targeted amplicon-based next-generation sequencing was performed at Quest Diagnostics using a DNA bait capture method on the NextSeq® (Illumina®) platform and the LeukoVantage® MDS Gene Panel, covering 36 MDS-related genes. The lower limit of sensitivity is 5% mutant alleles in the mixed population, and the percentage of mutant reads is reported and used to estimate the size of the clonal population.

[0218] Baseline mutation data were available for 165 of the 178 patients enrolled in the study (n=110 in the imetelstat group and n=55 in the placebo group). Mutated genes with a frequency of >10% in this study are listed in Table 26. [Table 26]

[0219] RBC-TI by number of baseline mutations: In patients with one or more detected mutations, imetelstat significantly improved RBC-TI response rates at 8 weeks (P=0.002) and 24 weeks (P<0.001) compared with placebo (Figure 24).

[0220] Significant rate differences were also observed in patients with more than two mutations at baseline: 45.5% vs. 6.7% for RBC-TI ≥8 weeks (P = 0.012) and 33.3% vs. 0% for RBC-TI ≥24 weeks (P = 0.014) for imetelstat vs. placebo, respectively.

[0221] Of the patients with mutation data, 161 (97.6%) had one or more mutations detected: 107 patients (97.3%) in the imetelstat group and 54 patients (98.2%) in the placebo group. The proportions of frequently occurring mutations were well balanced between treatment groups (Table 26).

[0222] SF3B1 mutations were detected at baseline in nearly 75% of patients in the imetelstat group and 78% of patients in the placebo group. Unfavorable prognostic mutations were identified in 20% of samples in both treatment groups, with two patients in each group harboring TP53 and two patients harboring RUNX1 mutations, and 18 vs. 6 patients in the imetelstat vs. placebo groups, respectively, with ASXL1 mutations, 2 vs. 1 patients harboring ETV6 mutations, and 0 vs. 2 patients harboring EZH2 mutations.

[0223] Among patients evaluated for commonly mutated genes in MDS, patients harboring SF3B1 mutations at baseline had significantly higher RBC-TI response rates with imetelstat versus placebo at both 8 weeks (48.8% vs. 16.3%; P=0.001) and 24 weeks (35.4% vs. 2.3%; P<0.001), as shown in Figure 22. Although the sample size was small, similar trends were seen in other commonly mutated genes (Figure 22) and in patients with SF3B1 hotspot mutations (≥2 patients in both groups) (Table 27). [Table 27]

[0224] RBC-TI rates by baseline mutation status of four sets of genes involved in different biological functions show that, consistent with the presence of a baseline SF3B1 mutation, patients with mutations in genes regulating the RNA spliceosome had significantly higher rates of 8+ week and 24+ week TI responses than placebo: 43.8% vs. 14.9% (P = 0.001) and 30.2% vs. 2.1% (P < 0.001), respectively (Figure 25). Similar trends were observed for other gene sets, although there were no significant differences between groups.

[0225] RBC-TI rates by baseline mutation status of poor prognostic genes show that imetelstat treatment demonstrated higher 8+ week (Table 28) and 24+ week RBC-TI response rates versus placebo in patients with poor prognostic genes mutated at baseline: 31.8% vs. 0%, and 9.1% vs. 0%, respectively. [Table 28]

[0226] These data indicate that, overall, imetelstat treatment resulted in higher TI rates than placebo in patients with diverse baseline mutation profiles. A significantly higher percentage of imetelstat-treated patients with baseline mutations in SF3B1, a gene commonly mutated in MDS and involved in regulating the RNA spliceosome, achieved RBC-TI at 8 and 24 weeks compared with placebo-treated patients. RBC-TI responses in imetelstat-treated patients occurred regardless of the presence or number of mutations associated with poor prognosis. Imetelstat-induced RBC-TI responses were observed across different molecularly defined subgroups, suggesting that the clinical benefit of imetelstat in LR-MDS patients is independent of the underlying molecular pattern.

[0227] A heatmap of the change in mutation burden in 18 patients with TI of 1 year or more is provided in Figure 23.

[0228] Example 4: Imetelstat produces clinically meaningful and statistically significant improvements in RBC-TI rates This is the first study to demonstrate a clinically meaningful and statistically significant improvement in RBC-TI rates in ESA-relapsed, refractory, or ineligible non-del(5q) LR-MDS patients treated with the telomerase inhibitor imetelstat compared with placebo. Among 8-week RBC-TI responders in the imetelstat group, RBC-TI was durable (median duration 52 weeks) and consistent over time, with 83% of patients experiencing a single, uninterrupted RBC-TI period. Twenty-eight and 18% of patients in the imetelstat group achieved RBC-TI for at least 24 weeks and 1 year, respectively. Significant improvements in RBC-TI were observed with imetelstat compared with placebo across different LR-MDS subgroups, including patients with and without RS and those with a high transfusion burden.

[0229] For the key secondary endpoint of HI-E, the difference in improvement rate between imetelstat and placebo based on the modified IWG 2006 criteria was not statistically significant. However, when HI-E was assessed using the more recent IWG 2018 criteria, which prioritize durability by measuring response for ≥16 weeks rather than ≥8 weeks, the effect of imetelstat was pronounced and more meaningful. Patients treated with imetelstat also demonstrated a significantly greater increase in hemoglobin levels over time, a biological measure that may be expected to translate to the clinical endpoint of RBC-TI.

[0230] These results also corresponded to the improvement in patient-reported fatigue in patients receiving imetelstat, with imetelstat-treated patients having a shorter median time to improvement and more likely to experience sustained, meaningful improvements in fatigue than placebo patients. This improvement in fatigue was not observed in other pivotal studies of treatments for LR-MDS, including luspatercept. The strong association between RBC-TI and sustained, meaningful improvements in fatigue further supports the clinical benefit of imetelstat treatment.

[0231] The mutation allele burden of SF3B1, TET2, ASXL1, and DNMT3A reflects the size of the malignant clone population and disease burden in LR-MDS. The maximum percentage reduction in mutant VAF was higher in patients treated with imetelstat compared to those treated with placebo, and VAF reduction correlated with RBC-TI rate, RBC-TI duration, and hemoglobin increase. Both clinical and molecular evidence indicate that imetelstat may restore functional erythropoiesis by reducing or eliminating the malignant clone, supporting the possibility of MDS disease modification.

[0232] Safety results were consistent with previous clinical experience with imetelstat, with no new safety signals reported. Dose delays and reductions to manage the common adverse events of neutropenia and thrombocytopenia maintained efficacy, suggesting that these events can be appropriately managed by treating physicians.

[0233] These cytopenias were the most common treatment-emergent AEs, with the majority occurring early in imetelstat treatment. Transient thrombocytopenia may represent an on-target effect of imetelstat treatment due to differential effects on malignant stem and progenitor cells and subsequent recovery of blood cell production. Importantly, due to their reversibility and short duration of onset, the clinical outcomes of grade 3-4 bleeding events, infections, and febrile neutropenia were similar in both treatment groups, suggesting no increased risk of these serious events.

[0234] One potential study limitation was that investigators were instructed to follow local clinical criteria for when patients should receive transfusions. Differences in local criteria may have led to variability in the sequelae prompting transfusions across the study. However, this variability was consistent across both arms and reflects the treatment of LR-MDS in real-world settings. In conclusion, imetelstat demonstrated durable RBC-TI, a significant reduction in transfusion burden, and an increase in hemoglobin levels in patients with LR-MDS who were relapsed, refractory, or ineligible for ESA treatment. Importantly, its robust activity, regardless of RS and mutation status and in patients with a high transfusion burden, distinguishes imetelstat from currently available therapies. Furthermore, durable RBC-TI for 24 weeks or even longer than 1 year has not previously been demonstrated. Improvements in fatigue, cytogenetic response, and reduction in VAF, as well as their correlation with clinical endpoints of transfusion dependence, support the disease-modifying potential of imetelstat. Taken together, the IMerge Phase 3 results confirm the observations from the Phase 2 portion and demonstrate that imetelstat provides significant clinical benefit in the severely TD LR-MDS patient population.

[0235] Example 5: Characteristics and clinical benefit of patients receiving imetelstat whose TI lasted for ≥1 year In the IMerge phase 3 trial, imetelstat produced higher TI rates of ≥8 weeks, ≥24 weeks, and ≥1 year (39.8%, 28.0%, and 17.8%) than placebo (15.0%, 3.3%, and 1.7%) in non-del(5q)LR-MDS patients who were RBC-TD, R / R / ineligible for ESAs, and not receiving lenalidomide or hypomethylating agents (HMAs). We report the characteristics and clinical benefit of patients whose TI persisted for ≥1 year from this study.

[0236] Results: Of 118 patients receiving imetelstat, 21 (17.8%, 95% CI [11.4%-25.9%]) achieved a TI lasting ≥1 year, representing 45% of TIs lasting ≥8 weeks (21 of 47 patients) and 64% of TIs lasting ≥24 weeks (21 of 33 patients). Of 60 patients receiving placebo and supportive care, 1 (1.7%) achieved a TI lasting ≥1 year. Among imetelstat responders with a TI lasting ≥1 year, 15 / 21 (71.4%) had ring sideroblasts, as did 1 placebo patient. The median prior RBC transfusion burden was 6 U (range, 4-9 U) over 8 weeks in the imetelstat group and 5 U in placebo patients. Central hemoglobin improved to a median of 5.2 g / dL in responders with ≥1 year of imetelstat treatment. Additional baseline characteristics are provided in Table 29. [Table 29]

[0237] Patients received imetelstat for a median of 101.1 weeks (range, 75.1-163.9 weeks) and a median of 24 cycles (range, 18-41 cycles). The median duration of imetelstat TI among responders with TI of 1 year or more was 123 weeks (95% CI, 80.4-not estimable), and the median increase in hemoglobin during the longest TI interval was 5.18 g / dL (range, 2.67-13.76 g / dL) in the imetelstat group compared with 1.67 g / dL in placebo patients. After a median follow-up of 125 weeks, none of the patients with TI of 1 year or more in either arm progressed to acute myeloid leukemia (AML). Of the patients receiving imetelstat, seven had abnormal karyotypes at baseline, six of whom achieved a reduction in the cytogenetic abnormality (four had a complete cytogenetic response and two had a partial cytogenetic response as determined by an independent review committee). Mutational data were available for 18 patients, all of whom had SF3B1 mutations at baseline, and multiple patients had concomitant TET2, DNMT3A, ASXL1, or JAK2 mutations. The maximum reduction in SF3B1 VAF in these patients ranged from -6% to -100%, and 13 of 18 (72.2%) achieved a VAF reduction of 50% or greater, including seven patients who completely eliminated VAF.

[0238] Reductions in other concurrent mutations were also observed in these patients. Safety was consistent with that previously reported, with the most frequent adverse events being reversible grade 3 or 4 thrombocytopenia and neutropenia. At the time of data cutoff, 13 patients receiving imetelstat and 13 patients receiving placebo were ongoing (Figure 21). Of the eight patients who discontinued treatment, seven lost response and one discontinued due to an adverse event. Analysis of progression-free survival and overall survival was not evaluable at this cutoff date due to insufficient follow-up.

[0239] Thus, treatment with imetelstat resulted in a sustained, uninterrupted TI of greater than 1 year in 17.8% of patients in the IMerge Phase 3 trial. In this ESA-R / R / ineligible population with a high prior transfusion burden, reduction to 0 RBC transfusions for greater than 1 year was associated with a reduced risk of transfusion-related complications, including end-organ dysfunction due to iron overload, and a reduced demand for healthcare resources. Furthermore, the durable TI and meaningful reduction in mutation burden suggest that imetelstat may have disease-modifying activity.

[0240] Example 6: Impact of MDS-associated mutations on the clinical efficacy of imetelstat To assess the impact of MDS-associated mutations on the clinical efficacy of imetelstat, we performed next-generation sequencing of a panel of 36 genes recurrently mutated in MDS. DNA sequencing was performed using peripheral blood samples collected at study entry. Further analysis of TI response to imetelstat was performed across various mutation subgroups defined based on genes involved in various biological functions, including splicing processes, epigenetic modifiers, transcriptional regulation, and receptor / kinases.

[0241] Baseline mutation data were available for 165 of 178 patients (imetelstat, n = 110; placebo, n = 55; 93.2% and 91.7% of the total in each group, respectively). Of patients with mutation data, 161 (97.6%) had one or more mutations detected, of which 75 (70.1%), 33 (30.8%), and 9 (8.4%) patients in the imetelstat group and 38 (70.4%), 15 (27.8%), and 7 (13%) patients in the placebo group had more than one, more than two, and more than three mutations, respectively. The 8-week or longer TI rates in the imetelstat vs. placebo groups were 42.7% vs. 15.8% (p=0.006) in patients with >1 mutation, 45.5% vs. 6.7% (p=0.012) in patients with >2 mutations, and 55.6% vs. 14.3% (p=0.089) in patients with >3 mutations. The 24-week or longer TI rates were 26.7% vs. 2.6% (p=0.003), 33.3% vs. 0% (p=0.014), and 33.3% vs. 0% (p=0.117), respectively.

[0242] Among patients with mutations associated with poor prognosis (TP53, ETV6, RUNX1, ASXL1, or EZH2), 31.8% and 9.1% of patients in the imetelstat group achieved a TI of 8 weeks or longer and 24 weeks or longer, respectively, compared with none in the placebo group. TP53 mutations were detected in two patients in each group; both patients in the imetelstat group achieved a TI of 8 weeks or longer, while none in the placebo group achieved a TI. Among patients with ASXL1 mutations, 5 of 18 patients (27.8%) in the imetelstat group and 0 of 6 patients (0) in the placebo group achieved a TI of 8 weeks or longer. Among patients with ETV6 mutations, 1 of 2 patients (50%) in the imetelstat group and 0 of 1 patient in the placebo group achieved a TI of 8 weeks or longer. Two patients in each group had RUNX1 mutations, and none achieved a TI.

[0243] Additional analyses of TI response were performed across four mutation subgroups defined based on genes involved in various biological functions, including: RNA spliceosome (e.g., SF3B1, U2AF1, SRSF2, and ZRSR2), epigenetic modifiers (e.g., TET2, DNMT3A, IDH1, IDH2, ASXL1, and EZH2), transcriptional regulation (e.g., RUNX1, BCOR, ETV6, SETBP1, GATA2, CEBPA, PHF6, NPM1, and STAT3), and receptor / kinase (e.g., CSF3R, FLT3, JAK2, KRAS, KIT, MPL, NRAS, and PTPN11). For patients with mutations in RNA spliceosome, epigenetic modifier, transcriptional regulation, and receptor / kinase, the 8-week TI rates with imetelstat were 43.8%, 37.7%, 40.0%, and 80.0%, respectively. The incidence of ≥24-week timeout was 30.2%, 27.5%, 20.0%, and 80.0%, respectively. The most frequently mutated gene was SF3B1 (125 of 165; 75.8%). The rates of ≥8-week timeout and ≥24-week timeout were 48.8% vs. 16.3% (p=0.001) and 35.4% vs. 2.3% (p<0.001) for imetelstat vs. placebo, respectively. In the imetelstat group, TI of ≥8 weeks was achieved in patients with a different spectrum of SF3B1 hotspot mutations: 2 of 8 patients (25.0%) with E622D, 4 of 7 patients (57.1%) with R625C / L / G, 7 of 12 patients (58.3%) with H662Q / N / D / Y, 2 of 2 patients (100%) with T663P, 2 of 6 patients (33.3%) with K666R / T / Q / N, 18 of 41 patients (43.9%) with K700E, 2 of 2 patients (100%) with A744P, and 1 of 2 patients (50.0%) with E783K. Durable TI of ≥24 weeks was also observed in patients with these hotspot mutations.

[0244] Other genes with mutation frequencies >10% were TET2 (32.7%), DNMT3A (17.0%), ASXL1 (14.5%), and CUX1 (12.7%). The ≥8-week TI rates in the imetelstat vs. placebo groups were 50% vs. 21.4% for TET2 mutations, 31.6% vs. 22.2% for DNMT3A mutations, 27.8% vs. 0% for ASXL1 mutations, and 35.7% vs. 14.3% for CUX1 mutations (Figure 22). The ≥24-week TI rates for these mutations were 40% vs. 0, 26.3% vs. 11.1%, 11.1% vs. 0, and 14.3% vs. 0, respectively (Figure 22).

[0245] Thus, in IMerge, higher RBC-TI rates were observed in patients with various baseline mutation profiles treated with imetelstat compared with placebo. TI responses in imetelstat-treated patients occurred regardless of the presence or number of mutations associated with poor prognosis. Imetelstat demonstrated comparable TI rates across different molecularly defined subgroups, suggesting that the clinical benefit of imetelstat in patients with LR-MDS is independent of the underlying molecular pattern.

[0246] Thus, in MDS patients, SF3B1 (involved in RNA splicing), TET2, DNMT3A, and ASXL1 (involved in epigenetic regulation) are commonly mutated genes, and quantification of these and other gene mutations can indicate disease burden and guide disease management.

[0247] In particular, a mechanistic link between the high prevalence of SF3B1 mutations and ringed sideroblasts in MDS has been established. Imetelstat is a first-in-class, direct, competitive inhibitor of telomerase activity that specifically targets the dysplastic clone, allowing for effective hematopoietic restoration.

[0248] As described herein, in the IMerge Phase 3 clinical trial of TI-dependent non-del(5q)LR-MDS patients who were relapsed / refractory / ineligible to ESAs, imetelstat demonstrated higher TI than placebo (15%, 3%, and 2%) for ≥8 weeks, ≥24 weeks, and ≥1 year (40%, 28%, and 18%).

[0249] Additionally, compared with placebo, treatment with imetelstat improved cytogenetic response rates, with a higher rate of patients achieving a 50% reduction in bone marrow RS cells (41% vs. 10%) and greater VAF reduction in the SF3B1, TET2, DNMT3A, and ASXL1 genes, which correlated with the clinical endpoints of RBC-TI response, longer duration of TI, and increased hemoglobin levels.

[0250] Example 7: Imetelstat treatment resulted in a sustained reduction in mutational burden over time Somatic point mutations are common in MDS. Some mutations strongly correlate with clinical phenotypic characteristics, including specific cytopenias, blast percentages, cytogenetic abnormalities, and overall survival in MDS patients. A reduction in mutant VAF may indicate a reduction in malignant clones and disease burden. As described herein, in the IMerge Phase 3 clinical trial of patients, the imetelstat group had a sustained reduction in SF3B1 VAF over time compared to the placebo group (Figure 18).

[0251] As shown by example in Figures 26A-C, three patients treated with imetelstat achieved a TI of more than 1 year, which correlated with a sustained decrease in VAF or elimination of mutant clones in multiple genes (e.g., SF3B1, DNMTA3, TET2, JAK2) during treatment. The time of increase in VAF and emergence of new mutations coincided with or preceded transfusion-dependent relapse. Placebo-treated patients had very limited effect on mutant VAF over time (Figures 27A-C).

[0252] Additionally, reduction of VAF in multiple genes by imetelstat correlated with TI response (Figures 19A and 19B), longer RBC-TI duration (Figure 12B, Figures 17A and 17B), and increased Hgb levels (Figure 12c, Figures 17C and 17D), suggesting that imetelstat may have the potential to modify the underlying biology of LR-MDS, reducing or eliminating the malignant clone and improving ineffective erythropoiesis.

[0253] Notwithstanding the full set of claims provided herein, the present disclosure provides the following enumerated provisions. Clause 1. A method for identifying a subject having myelodysplastic syndrome (MDS) for treatment with a telomerase inhibitor, the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A method wherein a 25% or greater reduction in VAF for one or more genes in a biological sample identifies subjects likely to benefit from treatment with a telomerase inhibitor. Clause 2. The method of clause 1, further comprising continuing treatment with a telomerase inhibitor if the biological sample obtained from the patient has a VAF for one or more genes that is reduced by 25% or more. Clause 3. The method of clause 1 or 2, wherein the subject is diagnosed with trisomy 8. Clause 4. The method of clause 3, wherein the subject is diagnosed with mosaic trisomy 8. Clause 5. The method of clause 3 or 4, further comprising diagnosing the subject with trisomy 8. Clause 6. A method of treating myelodysplastic syndrome (MDS), the method comprising: administering to a subject in need thereof an effective amount of a telomerase inhibitor; assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor. Clause 7. The method of clause 6, wherein the VAF for the one or more genes is reduced by 25% or more relative to the baseline VAF for the one or more genes prior to administration of the telomerase inhibitor. Clause 8. The method of clause 6 or 7, further comprising altering the dosage, dosage frequency, or course of treatment of the telomerase inhibitor administered to the subject based on the assessment. Clause 9. The method of any one of clauses 6 to 8, wherein the subject is diagnosed with trisomy 8. Clause 10. The method of clause 9, wherein the subject is diagnosed with mosaic trisomy 8. Clause 11. The method of clause 9 or 10, further comprising diagnosing the subject with trisomy 8. Clause 12. A method for monitoring therapeutic effectiveness in a subject having myelodysplastic syndrome (MDS), the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A method wherein a 25% or greater reduction in VAF for one or more genes in a biological sample identifies subjects likely to benefit from treatment with a telomerase inhibitor. Clause 13. The method of any one of clauses 1 to 12, wherein the subject has not been treated with an agent selected from a hypomethylating agent (HMA), lenalidomide, and combinations thereof. Clause 14. The method of any one of clauses 1 to 13, wherein the MDS is relapsed or refractory MDS. Clause 15. The method of any one of clauses 1 to 14, wherein the MDS is MDS that is relapsed or refractory to an erythropoiesis-stimulating agent (ESA). Clause 16. The method of any one of clauses 1 to 15, wherein the subject is classified as an IPSS low-risk or intermediate-1 risk MDS subject. Clause 17. The method of any one of clauses 1 to 16, wherein the subject is transfusion dependent. Clause 18. The method of clause 17, wherein the transfusion-dependent subject has a transfusion requirement of about 4 units or more during the 8 weeks prior to administration of the telomerase inhibitor. Clause 19. The method of any one of clauses 1 to 18, wherein the subject is a non-del5q human patient. Clause 20. The method of any one of clauses 1 to 19, wherein the subject has not been treated with lenalidomide. Clause 21. The method of any one of clauses 1 to 19, wherein the subject has not been treated with an HMA selected from decitabine and azacitidine. Clause 22. The method of any one of clauses 1 to 21, wherein the telomerase inhibitor is imetelstat. Clause 23. The method of clause 22, wherein the imetelstat is imetelstat sodium. Clause 24. The telomerase inhibitor is imetelstat and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (a) 7 to 10 mg / kg intravenous administration of imetelstat once every 4 weeks; (b) 7–10 mg / kg intravenous administration of imetelstat once weekly for 4 weeks; (c) intravenous administration of approximately 2.5 to 10 mg / kg of imetelstat once every 3 weeks; or (d) the method of clause 23, comprising intravenously administering 0.5 to 9.4 mg / kg of imetelstat once every four weeks. Clause 25. The method of clause 24, wherein each dosing cycle comprises intravenous administration of 7 to 10 mg / kg of imetelstat once every four weeks. Clause 26. The telomerase inhibitor is imetelstat sodium and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (a) 7 to 10 mg / kg intravenous administration of imetelstat sodium once every 4 weeks; (b) 7 to 10 mg / kg intravenous administration of imetelstat sodium once weekly for 4 weeks; (c) intravenous administration of approximately 2.5 to 10 mg / kg of imetelstat sodium once every 3 weeks; or (d) The method of clause 24, comprising intravenously administering 0.5 to 9.4 mg / kg of imetelstat sodium once every four weeks. Clause 27. The method of clause 26, wherein each dosing cycle comprises intravenous administration of about 4.5 to 11.7 mg / kg of imetelstat sodium once every four weeks. Clause 28. The method of clause 26, wherein each dosing cycle comprises intravenous administration of about 7.5 mg / kg of imetelstat sodium once every four weeks. Clause 29. A method for identifying a subject having myelodysplastic syndrome (MDS) for treatment with a telomerase inhibitor, the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A method wherein a 25% or greater reduction in VAF for one or more genes in a biological sample identifies subjects likely to benefit from treatment with a telomerase inhibitor. Clause 30. The method of clause 29, further comprising continuing treatment with a telomerase inhibitor if the biological sample obtained from the patient has a VAF for one or more genes that is reduced by 25% or more. Clause 31. The method of clause 29 or 30, wherein the subject is diagnosed with trisomy 8. Clause 32. The method of clause 31, wherein the subject is diagnosed with mosaic trisomy 8. Clause 33. The method of clause 31 or 32, further comprising diagnosing the subject with trisomy 8. Clause 34. A method of treating myelodysplastic syndrome (MDS), comprising: administering to a subject in need thereof an effective amount of a telomerase inhibitor; assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor. Clause 35. The method of clause 34, wherein the VAF for the one or more genes is reduced by 25% or more relative to the baseline VAF for the one or more genes prior to administration of the telomerase inhibitor. Clause 36. The method of clause 34 or 35, further comprising altering the dosage, dosage frequency, or course of treatment of the telomerase inhibitor administered to the subject based on the assessment. Clause 37. The method of any one of clauses 34 to 36, wherein the subject is diagnosed with trisomy 8. Clause 38. The method of clause 37, wherein the subject is diagnosed with mosaic trisomy 8. Clause 39. The method of clause 37 or 38, further comprising diagnosing the subject as having trisomy 8. Clause 40. A method for monitoring therapeutic efficacy in a subject with myelodysplastic syndrome (MDS), the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A method wherein a 25% or greater reduction in VAF for one or more genes in a biological sample identifies subjects likely to benefit from treatment with a telomerase inhibitor. Clause 41. The method of any one of clauses 29 to 40, wherein the subject has not been treated with an agent selected from a hypomethylating agent (HMA), lenalidomide, and combinations thereof. Clause 42. The method of any one of clauses 29 to 41, wherein the MDS is relapsed or refractory MDS. Clause 43. The method of any one of clauses 29 to 42, wherein the MDS is MDS that is relapsed or refractory to an erythropoiesis-stimulating agent (ESA). Clause 44. The method of any one of clauses 29 to 43, wherein the subject is classified as an IPSS low-risk or intermediate-1 risk MDS subject. Clause 45. The method of any one of clauses 29 to 44, wherein the subject is transfusion dependent. Clause 46. The method of clause 45, wherein the transfusion-dependent subject has a transfusion requirement of about 4 units or more during the 8 weeks prior to administration of the telomerase inhibitor. Clause 47. The method of any one of clauses 29 to 46, wherein the subject is a non-del5q human patient. Clause 48. The method of any one of clauses 29 to 47, wherein the subject is not undergoing treatment with lenalidomide. Clause 49. The method of any one of clauses 29 to 48, wherein the subject has not been treated with an HMA selected from decitabine and azacitidine. Clause 50. The method of any one of clauses 29 to 49, wherein the telomerase inhibitor is imetelstat. Clause 51. The method according to clause 50, wherein the imetelstat is imetelstat sodium. Clause 52. The telomerase inhibitor is imetelstat and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (e) 7 to 10 mg / kg intravenous administration of imetelstat once every 4 weeks; (f) 7 to 10 mg / kg intravenous administration of imetelstat once weekly for 4 weeks; (g) 2.5 to 10 mg / kg intravenous administration of imetelstat once every 3 weeks; or (h) The method of clause 51, comprising intravenously administering 0.5 to 9.4 mg / kg of imetelstat once every four weeks. Clause 53. The method of clause 52, wherein each dosing cycle comprises intravenous administration of 7 to 10 mg / kg of imetelstat once every four weeks. Clause 54. The telomerase inhibitor is imetelstat sodium and is administered over 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (e) 7 to 10 mg / kg intravenous administration of imetelstat sodium once every 4 weeks; (f) 7 to 10 mg / kg intravenous administration of imetelstat sodium once weekly for 4 weeks; (g) 2.5 to 10 mg / kg intravenous administration of imetelstat sodium once every 3 weeks, or (h) The method of clause 52, comprising intravenously administering 0.5 to 9.4 mg / kg of imetelstat sodium once every four weeks. Clause 55. The method of clause 54, wherein each dosing cycle comprises intravenous administration of about 4.5 to 11.7 mg / kg of imetelstat sodium once every four weeks. Clause 56. The method of clause 54, wherein each dosing cycle comprises intravenous administration of about 7.5 mg / kg of imetelstat sodium once every four weeks.

[0254] Although particular embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent that certain changes and modifications may be made in light of the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0255] Thus, the foregoing merely illustrates the principles of the invention. Various arrangements can be devised that embody the principles of the invention and are within its spirit and scope, although not explicitly described or shown herein. Moreover, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to advance the art, and should be construed as including no limitations to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, regardless of structure, i.e., any elements developed to perform the same function. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are specifically indicated by the appended claims.

Claims

1. 1. A method for identifying a subject having myelodysplastic syndrome (MDS) for treatment with a telomerase inhibitor, the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject after administration of the telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A reduction of 25% or more in the VAF for the one or more genes in the biological sample identifies a subject who is likely to benefit from treatment with the telomerase inhibitor.

2. 10. The method of claim 1, further comprising continuing the treatment with the telomerase inhibitor if the biological sample obtained from the subject has the VAF for the one or more genes reduced by 25% or more.

3. 3. The method of claim 1 or 2, wherein the subject is diagnosed with trisomy 8.

4. 4. The method of claim 3, wherein the subject is diagnosed with mosaic trisomy 8.

5. 5. The method of claim 3 or 4, further comprising diagnosing the subject as having trisomy 8.

6. 1. A method of treating myelodysplastic syndrome (MDS), said method comprising: administering to a subject in need thereof an effective amount of a telomerase inhibitor; assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject after administration of the telomerase inhibitor.

7. 7. The method of claim 6, wherein the VAF for the one or more genes is reduced by 25% or more relative to a baseline VAF for the one or more genes prior to administration of the telomerase inhibitor.

8. 8. The method of claim 6 or 7, further comprising altering the dosage, dosage frequency, or course of therapy of the telomerase inhibitor administered to the subject based on the evaluation.

9. The method of any one of claims 6 to 8, wherein the subject is diagnosed with trisomy 8.

10. 10. The method of claim 9, wherein the subject is diagnosed with mosaic trisomy 8.

11. 11. The method of claim 9 or 10, further comprising diagnosing the subject as having trisomy 8.

12. 1. A method for monitoring therapeutic effectiveness in a subject having myelodysplastic syndrome (MDS), the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A reduction of 25% or more in the VAF for the one or more genes in the biological sample identifies a subject who is likely to benefit from treatment with the telomerase inhibitor.

13. 13. The method of any one of claims 1 to 12, wherein the subject has not been treated with an agent selected from a hypomethylating agent (HMA), lenalidomide, and combinations thereof.

14. The method of any one of claims 1 to 13, wherein the MDS is relapsed or refractory MDS.

15. The method of any one of claims 1 to 14, wherein the MDS is relapsed or refractory to an erythropoiesis-stimulating agent (ESA).

16. 16. The method of any one of claims 1-15, wherein the subject is classified as an IPSS low risk or intermediate 1 risk MDS subject.

17. The method of any one of claims 1 to 16, wherein the subject is transfusion dependent.

18. 18. The method of claim 17, wherein the transfusion-dependent subject has a transfusion requirement of about 4 units or more during the 8 weeks prior to the administration of the telomerase inhibitor.

19. The method of any one of claims 1 to 18, wherein the subject is a non-del5q human patient.

20. 20. The method of any one of claims 1 to 19, wherein the subject has not been treated with lenalidomide.

21. 20. The method of any one of claims 1 to 19, wherein the subject has not been treated with an HMA selected from decitabine and azacitidine.

22. The method of any one of claims 1 to 21, wherein the telomerase inhibitor is imetelstat.

23. 23. The method of claim 22, wherein the imetelstat is imetelstat sodium.

24. The telomerase inhibitor is imetelstat and is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (a) 7-10 mg / kg of imetelstat administered intravenously once every four weeks; (b) 7-10 mg / kg of imetelstat administered intravenously once weekly for 4 weeks; (c) 2.5 to 10 mg / kg of imetelstat administered intravenously once every three weeks; or 24. The method of claim 23, comprising (d) intravenous administration of 0.5 to 9.4 mg / kg of imetelstat once every four weeks.

25. 25. The method of claim 24, wherein each dosing cycle comprises intravenous administration of 7-10 mg / kg of imetelstat once every four weeks.

26. The telomerase inhibitor is imetelstat sodium and is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (a) intravenous administration of 7 to 10 mg / kg of imetelstat sodium once every 4 weeks; (b) 7-10 mg / kg of imetelstat sodium administered intravenously once a week for 4 weeks; (c) 2.5 to 10 mg / kg of imetelstat sodium administered intravenously once every 3 weeks; or 25. The method of claim 24, comprising (d) intravenously administering 0.5 to 9.4 mg / kg of imetelstat sodium once every four weeks.

27. 27. The method of claim 26, wherein each dosing cycle comprises intravenous administration of 4.5 to 11.7 mg / kg of imetelstat sodium once every four weeks.

28. 27. The method of claim 26, wherein each dosing cycle comprises intravenous administration of 7.5 mg / kg of imetelstat sodium once every four weeks.

29. 1. A method for identifying a subject having myelodysplastic syndrome (MDS) for treatment with a telomerase inhibitor, the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject after administration of the telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A reduction of 25% or more in the VAF for the one or more genes in the biological sample identifies a subject who is likely to benefit from treatment with the telomerase inhibitor.

30. 30. The method of claim 29, further comprising continuing the treatment with the telomerase inhibitor if the biological sample obtained from the subject has the VAF for the one or more genes reduced by 25% or more.

31. 31. The method of claim 29 or 30, wherein the subject is diagnosed with trisomy 8.

32. 32. The method of claim 31 , wherein the subject is diagnosed with mosaic trisomy 8.

33. 33. The method of claim 31 or 32, further comprising diagnosing the subject as having trisomy 8.

34. 1. A method of treating myelodysplastic syndrome (MDS), said method comprising: administering to a subject in need thereof an effective amount of a telomerase inhibitor; assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject after administration of the telomerase inhibitor.

35. 35. The method of claim 34, wherein the VAF for the one or more genes is reduced by 25% or more relative to a baseline VAF for the one or more genes prior to administration of the telomerase inhibitor.

36. 36. The method of claim 34 or 35, further comprising altering the dosage, dosage frequency, or course of therapy of the telomerase inhibitor administered to the subject based on the evaluation.

37. 37. The method of any one of claims 34 to 36, wherein the subject is diagnosed with trisomy 8.

38. 38. The method of claim 37, wherein the subject is diagnosed with mosaic trisomy 8.

39. 39. The method of claim 37 or 38, further comprising diagnosing the subject as having trisomy 8.

40. 1. A method for monitoring therapeutic effectiveness in a subject having myelodysplastic syndrome (MDS), the method comprising: assessing the variant allele frequency (VAF) for one or more genes selected from the group consisting of SF3B1, TET2, DNMT3A, CUX1, and ASXL1 in a biological sample obtained from the subject after administration of a telomerase inhibitor; comparing the VAF for the one or more genes to a baseline VAF for the one or more genes before administration of the telomerase inhibitor; A reduction of 25% or more in the VAF for the one or more genes in the biological sample identifies a subject who is likely to benefit from treatment with the telomerase inhibitor.

41. 41. The method of any one of claims 29-40, wherein the subject has not been treated with an agent selected from a hypomethylating agent (HMA), lenalidomide, and combinations thereof.

42. The method of any one of claims 29 to 41, wherein the MDS is relapsed or refractory MDS.

43. 43. The method of any one of claims 29 to 42, wherein the MDS is relapsed or refractory to an erythropoiesis-stimulating agent (ESA).

44. 44. The method of any one of claims 29-43, wherein the subject is classified as an IPSS low risk or intermediate 1 risk MDS subject.

45. 45. The method of any one of claims 29 to 44, wherein the subject is transfusion dependent.

46. 46. ​​The method of claim 45, wherein the transfusion-dependent subject has a transfusion requirement of about 4 units or more during the 8 weeks prior to the administration of the telomerase inhibitor.

47. The method of any one of claims 29 to 46, wherein the subject is a non-del5q human patient.

48. 48. The method of any one of claims 29 to 47, wherein the subject has not been treated with lenalidomide.

49. 49. The method of any one of claims 29 to 48, wherein the subject has not been treated with an HMA selected from decitabine and azacitidine.

50. 50. The method of any one of claims 29 to 49, wherein the telomerase inhibitor is imetelstat.

51. 51. The method of claim 50, wherein the imetelstat is imetelstat sodium.

52. The telomerase inhibitor is imetelstat and is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (e) 7-10 mg / kg of imetelstat administered intravenously once every 4 weeks; (f) 7-10 mg / kg of imetelstat administered intravenously once a week for 4 weeks; (g) 2.5 to 10 mg / kg of imetelstat administered intravenously once every three weeks; or 52. The method of claim 51, comprising (h) intravenous administration of 0.5 to 9.4 mg / kg of imetelstat once every four weeks.

53. 53. The method of claim 52, wherein each dosing cycle comprises intravenous administration of 7-10 mg / kg of imetelstat once every four weeks.

54. The telomerase inhibitor is imetelstat sodium and is administered for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each cycle comprising: (e) 7-10 mg / kg intravenous administration of imetelstat sodium once every 4 weeks; (f) 7-10 mg / kg of imetelstat sodium administered intravenously once a week for 4 weeks; (g) 2.5 to 10 mg / kg of imetelstat sodium administered intravenously once every three weeks; or 53. The method of claim 52, comprising (h) intravenously administering 0.5 to 9.4 mg / kg of imetelstat sodium once every four weeks.

55. 55. The method of claim 54, wherein each dosing cycle comprises intravenous administration of 4.5 to 11.7 mg / kg of imetelstat sodium once every four weeks.

56. 55. The method of claim 54, wherein each dosing cycle comprises intravenous administration of 7.5 mg / kg of imetelstat sodium once every four weeks.

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