Imetelstat-mediated alterations in fatty acid metabolism to induce ferroptosis as a therapeutic strategy for acute myeloid leukemia.

Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft resource and perform integrated genomics, transcriptomics and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized phase II-like preclinical trial in patient-derived xenografts, imetelstat effectively diminishes AML burden and preferentially targets subgroups containing mutant NRAS and oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing substantial disease control in AML.

R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer. SIGNIFICANCE: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.

Oncogenic drivers dictate immune control of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.

Hematopoietic stem and progenitor cell-restricted Cdx2 expression induces transformation to myelodysplasia and acute leukemia.

The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs). Cdx2-transgenic mice develop myelodysplastic syndrome with progression to acute leukemia associated with acquisition of additional driver mutations. Cdx2-expressing HSPCs demonstrate enrichment of hematopoietic-specific enhancers associated with pro-differentiation transcription factors. Furthermore, treatment of Cdx2 AML with azacitidine decreases leukemic burden. Extended scheduling of low-dose azacitidine shows greater efficacy in comparison to intermittent higher-dose azacitidine, linked to more specific epigenetic modulation. Conditional Cdx2 expression in HSPCs is an inducible model of de novo leukemic transformation and can be used to optimize treatment in high-risk AML.

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.

[Vascular endothelial injury induced by anti-endothelial cell antibody in allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To clarify the role of endothelial cells (ECs) injury induced by anti-endothelial cell antibody (AECA) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Serum immunoglobulin (IgG) from allo-HSCT recipients were purified and incubated with human umbilical vein vascular endothelium (HUVEC) in vitro, then the functional changes and cell apoptosis were tested. RESULTS: After incubation with AECA positive IgG, soluble adhesion molecules significantly elevated in culture supernatant. When concentration of IgG was 160, 320, and 640 µg/ml, concentrations of soluble intercellular adhesion molecule-1 in supernatant were statistically higher in AECA positive groups [(117.10 ± 12.82) vs (78.17 ± 4.90) pg/ml, (151.30 ± 15.35) vs (89.46 ± 6.02) pg/ml, (239.00 ± 32.53) vs (127.80 ± 13.86) pg/ml, P<0.01)]. When concentration of IgG was 40, 80, 160, 320, and 640 µg/ml, concentrations of soluble vascular cell adhesion molecule-1 in supernatant were also statistically higher in AECA positive groups [(38.51 ± 3.76) vs (24.78 ± 2.59) pg/ml, (61.34 ± 6.99) vs (38.20 ± 3.17) pg/ml, (135.60 ± 24.46) vs (63.73 ± 5.08) pg/ml, (221.30 ± 29.40) vs (112.80 ± 8.91) pg/ml, (420.90 ± 31.70) vs (224.40 ± 20.79) pg/ml, P<0.01]. Clotting activity factors also elevated in culture supernatant after incubation with AECA positive IgG. When concentration of IgG was 80, 160, 320, and 640 µg/ml, concentrations of von Willebrand factor were statistically higher in AECA positive groups [(19.51 ± 0.72) vs (17.17 ± 0.60) ng/ml, P=0.0193; (22.97 ± 1.18) vs (18.27 ± 0.61) ng/ml, (26.40 ± 1.54) vs (19.53 ± 0.70) ng/ml, (34.35 ± 1.60) vs (23.81 ± 0.92) ng/ml, P<0.01]. When concentration of IgG was 320 and 640 µg/ml, concentrations of thrombomodulin were statistically higher in AECA positive groups [(57.50 ± 4.50) vs (40.31 ± 4.39) pg/ml, P=0.0132; (59.18 ± 4.11) vs (38.84 ± 5.16) pg/ml, P<0.01]. However, inflammatory factors (IL-1ß, IL-6, IL-8 and ANG2) were not statistically different in AECA positive and negative groups (P>0.05). Moreover, IgG from AECA positive samples did not change the proliferation or cell apoptosis. CONCLUSION: AECA from allo-HSCT recipients dysregulates ECs' function in vitro, but do not induce apoptosis, which is valuable in the pathophysiology of graft-versus-host disease (GVHD) and other complications after allo-HSCT.

Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy.

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc(-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs.

[IL-18 single nucleotide polymorphisms in hematologic malignancies with HLA matched sibling donor allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the impact of interleukin-18 (IL-18) single nucleotide polymorphisms on outcomes of hematologic malignancies with HLA-matched sibling donor hematopoietic stem cell transplantation (allo-HSCT). METHODS: Single- nucleotide polymorphisms in IL-18 promoter was detected by PCR-sequence-specific primer analysis (PCR-SSP) in 93 recipients and their HLA matched sibling donors. Hematopoietic reconstitution, incidences of graft versus host disease (GVHD) and infections, transplant related mortality (TRM), and disease free survival (DFS) were analyzed. RESULTS: In comparison with -137 G/C+C/C donor genotype, patients with -137 G/G donor genotype had shorter duration of neutrophil recovery [15(11-23) days vs 17(11-24) days, P=0.01], higher incidence of extensive chronic GVHD (20.6% vs 3.3%, P=0.029), but no difference in the interval of platelet recovery [20(11-46) days vs 20(7-38) days, P=0.844]. The incidence of extensive chronic GVHD in -607 C/C donor genotype (31.6%) was significantly higher than that (10.8%) in C/A + A/A donor genotype (P=0.024). Recipients with -607 C/C genotype also had higher incidence (33.3%) of extensive chronic GVHD than those with C/A+A/A genotype (10.7%, P=0.016). There were no differences in acute GVHD, TRM, and DFS between different genotypes. CONCLUSION: IL-18 -137 G homozygous genotype in donor facilitated neutrophil reconstitution, but increased the risk of extensive chronic GVHD in patients with allo-HSCT.

Clinical significance of anti-endothelial cell antibody in allogeneic hematopoietic stem cell transplantation recipients with graft-versus-host disease.

Anti-endothelial cell antibody (AECA) is well known to reflect endothelial injury. Graft-versus-host disease (GVHD), a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), is also closely associated with endothelial injury. We hypothesized that AECA may be associated with GVHD. To investigate the clinical significance of AECA in allo-HSCT recipients with GVHD, we detected AECA by cyto-enzyme-linked immunosorbent assay (cyto-ELISA) in allo-HSCT recipients with acute and/or chronic GVHD (aGVHD and cGVHD). Incidences of anti-HMEC-1 AECA (anti-HMEC) and anti-EA.hy926 AECA (anti-EAHY) were significantly higher in patients with grade II-IV than grade 0-I aGVHD (P = 0.049, P = 0.011, respectively). There was no difference in the incidence of AECA between patients with and without cGVHD. Patients with anti-EAHY positive in the early stage post-transplant demonstrated a higher incidence of cGVHD (P = 0.044). In patients with grade 0-I aGVHD, AECA-positive patients had higher overall survival and disease-free survival (P < 0.05), and tended to have lower incidences of relapse and transplant-related mortality. Our data suggest that AECA plays an important role in the pathogenesis of GVHD.

Epidemiology and risk factors for chronic kidney disease in patients with ischaemic stroke.

BACKGROUND: There is growing evidence for an association between chronic renal disease (CKD) and adverse cerebrovascular events because of the overlap of several risk factors. The purpose of this study is to examine the epidemiology of CKD and the characteristics of risk factors for CKD in the population with ischaemic stroke. METHODS: This retrospective study included 571 patients with ischaemic stroke. Estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease (MDRD) study equation. Renal function was assessed according to the Kidney Disease Outcomes Quality Initiative (K/DOQI)-CKD classification. RESULTS: Study demonstrated that the major factors associated with CKD in the ischaemic stroke patients were age, diabetes mellitus, hypertension, systolic blood pressure, LDL cholesterol and serum uric acid. Diabetes mellitus (OR 4·146, 95% CI 1·047-16·418, P = 0·043), hypertension and diabetes mellitus (OR 3·574, 95% CI 1·248-10·234, P = 0·018), serum uric acid (OR 1·010, 95% CI 1·006-1·013, P < 0·001) and LDL cholesterol (OR 1·431, 95% CI 1·063-1·928, P = 0·018) were independent risk factors associated with CKD in the patients with ischaemic stroke. CONCLUSIONS: The patients with ischaemic stroke may be considered as a high-risk population for CKD and be aggressively managed for CKD prevention. The high prevalence of CKD in population with ischaemic stroke prompts the need for greater public awareness about risks of CKD.

[Role of IFN-γ + 874 genetic polymorphisms in allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the impact of IFN-γ + 874 polymorphisms on the outcome in HLA matched sibling HSCT. METHODS: We used PCR-sequence-specific primer analysis (PCR-SSP) to analyze the polymorphisms of IFN-γ + 874 T/A in 80 recipient and donor pairs from October 2005 to March 2008. RESULTS: Recipients having donors who possessed IFN-γ + 874 A/A genotype had significantly earlier neutrophil recovery compared with those having donors with non-A/A genotype (15 (11 - 27) days vs 18 (12 - 30) days, P = 0.029). And IFN-γ + 874 A/A in both recipients and donors further facilitated neutrophil recovery compared with others (13 (11 - 25) days and 19 (12 - 31) days, P = 0.019). Besides, IFN-γ + 874 A/A in recipients increased the probability of grade II-IV acute graft versus disease (aGVHD) and cytomegalovirus viraemia compared with IFN-γ + 874 T/A or T/T genotype (20% vs 4% P = 0.041, 43.6% vs 16.0% P = 0.032), which lead to increased 5-year transplant-related mortality (TRM) (33.7% ± 6.8% vs 12.0% ± 6.5%, P = 0.050) and decreased 5-year event free survival (EFS) \[(58.2 ± 6.7)% vs (84.0 ± 7.3)%, P = 0.032\] compared with the latter. IFN-γ + 874 A/A in both recipients and donors also significantly increased the probability of grade II-IV aGVHD and cytomegalovirus viraemia compared with the other (21.7% vs 5.9%, P = 0.050; 45.7% vs 20.6%, P = 0.020), which caused increased 5-year TRM \[(31.6 ± 7.5)% vs (13.6 ± 6.5)%, P = 0.048\] and decreased 5-year EFS \[(56.8 ± 7.3)% vs (79.4 ± 6.9)%, P = 0.037\] compared with the other. CONCLUSION: In HLA-matched sibling HSCT setting, the presence of IFN-γ + 874 T allele in recipients or in both recipients and donors significantly decreased the risk of grade II-IV aGVHD and CMV infection and increased EFS. While IFN-γ + 874 A/A in donors or in both recipients and donors was associated with shorter duration to neutrophil recovery.

[Preliminary analysis of therapeutic efficacy and prognosis of allogeneic hematopoietic stem cell transplantation in patients with advanced chronic myeloid leukemia].

Chronic myeloid leukemia (CML) at advanced and blastic phase is a disease with poor prognosis, for which allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only treatment choice with curative potential. This study was purposed to investigate the therapeutic efficacy of allo-HSCT and prognosis of advanced CML patients. The 28 cases of CML in accelerated phase or blast crisis received allo-HSCT were analysed retrospectively in terms curative efficacy, basic characteristics before transplant and prognosis, therapeutic strategy before transplant and prognosis, events after transplant and prognosis. The results indicated that 10 out of 28 patients were in complete remission, showing a 3-year overall survival and disease-free survival rate of 34.9% and 35.7% respectively; 18 patients died. Univariate analysis revealed that the clonal evolution and blast amount are baseline risk factor of poor prognosis, and combination of them can be used to predict the outcome of patients; application of imatinib before transplant and achievement of complete hematologic remission could not improve the prognosis; severe aGVHD among post-transplant events was proven to be a negative prognostic factor. It is concluded that for advanced CML patients received allo-HSCT, clonal evolution and blast percentage are prognostic factors, and the pre-transplant use of imatinib did not influence the outcome.

[Risk factors and prognosis of invasive fungal infections in patients with hematological diseases].

OBJECTIVE: To investigate the incidence, risk factors, prognosis and high risk patients of invasive fungal infections (IFI) in patients with hematological diseases. METHODS: : Over 2-week hospitalized patients from January 2007 to December 2008 were retrospectively reviewed. Logistic regression was used to analyze the risk factors of IFI, and recursive partitioning to reveal high risk patients. Incidence of IFI was estimated by cumulative incidence function, and the prognosis by Kaplan-Meier method. RESULTS: A total of 1048 assessable treatment cycles were recorded and 93 cases of IFI were diagnosed, with an incidence of 8.87 per 100 treatment cycles. Multivariate logistic regression revealed the following risk factors: age (OR 1.025, 95% CI 1.010-1.041, P = 0.002), duration of neutropenia (OR 1.028, 95% CI 1.014-1.042, P < 0.0001) and uncontrolled underlying diseases (OR 2.620, 95% CI 1.608-4.268, P = 0.0001). Recursive partitioning found two groups of high risk patients: (1) patients with uncontrolled underlying diseases and neutropenia duration > or = 58 days (7/12, 58.3%), (2) patients with uncontrolled underlying diseases and age > or = 33 years (40/208, 19.2%). At the end of follow-up, 111 cases of IFI were recorded in 451 patients, with a 1-year cumulative incidence of 27.1%. In patients with established IFI, overall survival rate and IFI related mortality rate at 12 weeks after diagnosis were 83.4% and 13.5% respectively. CONCLUSION: Age, duration of neutropenia and uncontrolled underlying diseases are risk factors of IFI; patients with uncontrolled underlying diseases and age > or = 33 years were at high risk of IFI and need major concern. IFI has a better prognosis and a lower related mortality in this study.

Secondary antifungal prophylaxis in hematological malignancies in a tertiary medical center.

To investigate the efficacy of secondary antifungal prophylaxis (SAP) in patients with hematological diseases, all medical records within two consecutive years were retrospectively reviewed. In all, 57 patients with a history of invasive fungal infections received 149 cycles of further therapy for their underlying hematological diseases. Logistic regression and recursive partitioning were used to discriminate high risk patients for failure of SAP. After a median follow-up period of 120 (12-1,080) days, 11 cases (7.4/100 cycles) experienced failure of SAP, including 5 cases during allo-SCT and 6 cases during chemotherapy, corresponding to cumulative incidence at 24.5%. Multivariate logistic regression revealed two risk factors for failure of SAP: use of high dose corticosteroid (OR 13.5, 95% CI 3.09-58.6, P = 0.0005) and duration of neutropenia ≥ 14 days (OR 7.47, 95% CI 1.69-32.9, P = 0.008). Recursive partitioning found that patients with these two risk factors were in high risk, with SAP failure rate as high as 50.0%. In conclusion, use of high dose corticosteroid and duration of neutropenia ≥ 14 days were risk factors of SAP failure. Patients with the two risk factors concurrently were in high risk and needed special concern.

[Allogeneic stem cell transplantation for 75 cases of acute myeloid leukemia in complete remission: outcome and prognostic analysis].

This study was purposed to evaluate the outcome of patients with acute myeloid leukemia (AML) who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in complete remission, and to study the prognostic factors. 75 cases of AML in complete remission receiving allo-HSCT from January 2000 to December 2007 were retrospectively analyzed. Major end points of study included overall survival (OS), disease free survival (DFS), relapse rate and transplantation related mortality (TRM). The results showed that 3-year OS and DFS of the study population reached to 58.4% and 53.9% respectively, and the relapse rate and TRM leaded to 16.9% and 29.9% respectively. Incidence of acute GVHD was 59.6%, with 18.7% II-IV aGVHD. Different prognosis was observed between HSCT recipients of alternative donor and HLA-matched related donor (MRD) (3-year DFS was 34.3% vs 60.0%, p = 0.019), between patients of refractory leukemia and the control (3-year DFS was 35.7% vs 58.2%, p = 0.048), between recipients with and without severe aGVHD (3-year DFS was 35.7% vs 54.4%, p = 0.059). Further analysis revealed significantly high TRM in recipients receiving allo-HSCT of alternative donor (p = 0.033) and high rate of severe aGVHD (p = 0.010). Multivariate analysis revealed three negative prognostic factors: donor availability (alternative vs MRD) (p = 0.049, RR = 2.09, 95%CI 1.01 - 4.36), refractory leukemia (p = 0.038, RR = 2.33, 95%CI 1.05 - 5.20) and severe aGVHD (p = 0.040, RR = 2.33, 95%CI 1.04 - 5.20). It is concluded that allo-HSCT is a choice for the AML case at complete remission and TRM is the major cause of the transplantation failure. Donor availability, refractory leukemia and severe aGVHD are confirmed as risk factors of poor prognosis for allo-HSCT patients with AML in CR.

[Allogeneic hematopoietic stem cell transplantation for 16 patients with aplastic anemia].

OBJECTIVE: To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aplastic anemia (AA). METHODS: Twelve patients with severe AA (SAA) and 4 with chronic AA (CAA) received allo- HSCT. The effectiveness and complication were analyzed retrospectively. RESULTS: Hematopoiesis reconstitution was achieved 14 patients (87.50%). The median time of neutrophils reached to 0.5 x 10(9)/L and platelets reached to 20 x 10(9)/L were 14 (11 - 16) and 14 (10 - 33) days, respectively. Six cases developed grade I - II acute graft-versus-host disease (aGVHD), chronic local GVHD occurred in 2 patients. Graft rejection occurred in 3 cases. Thirteen cases survived with a median of 10 (0.5 - 84) months at the end of follow-up. Three cases died of un-engraftment, graft rejection (GR) and interstitial pneumonia (IP) each. CONCLUSION: Allo-HSCT is an effective therapy for patients with AA. Enhancing immunosuppressive treatment for conditioning and GVHD prophylaxis may reduce the incidence of GR and GVHD.