Survival In Leukemia Research Articles

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Real world analysis on the determinants of survival in primary plasma cell leukemia in the United States.

Real world analysis on the determinants of survival in primary plasma cell leukemia in the United States.

Acute myeloid leukemia-derived bone marrow mesenchymal cells exhibit improved support for leukemic cell proliferation

IntroductionThe bone marrow (BM) microenvironment plays a significant role in acute myeloid leukemia (AML) genesis and there is evidence that BM mesenchymal stromal cells (BMMSCs) can support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. Hypothesis and methodNevertheless, currently unknown are the relevance of the spatial localization of AML cells relative to the BMMSCs and whether BMMSCs from patients with AML and healthy subjects have similar properties. To address these issues, we performed a differential gene expression analysis using RNA-sequencing data generated from healthy donors (HDs) and leukemic BMMSCs. ResultsThe Gene Set Enrichment Analysis (GSEA) revealed that leukemic BMMSCs were associated with the terms “positive regulation of cell cycle”, “angiogenesis” and “signaling by the estimated glomerular filtration rate (eGFR)”, whereas healthy donor (HD)-derived BMMSCs were associated with “programmed cell death in response to the reactive oxygen species (ROS)”, “negative regulation of the cytochrome C from the mitochondria” and “interferon signaling”. Next, we evaluated the mitochondrial superoxide production in AML cells in a co-culture layered model. The superoxide production was reduced in leukemic cells in close contact (adhered to the surface or beneath the cell layer) with BMMSCs, indicating lower oxidative stress. ConclusionTaken together, our results suggest that AML-derived BMMSCs are transcriptionally rewired and can reduce the metabolic stress of leukemic cells.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

Incidence-Based Mortality (IBM) and Survival for Chronic Myeloid Leukemia (CML) By Age Group: Is There Any Relationship between Age of Diagnosis, IBM and Survival?

Incidence-Based Mortality (IBM) and Survival for Chronic Myeloid Leukemia (CML) by Age group: Is there any relationship between age of diagnosis, IBM and Survival? Background: CML is known to affect different age groups differently, with a predominance in adults. CML accounts for about 15% of all new cases of leukemia, with over 50% of the cases being diagnosed in individuals 65 or older. Our study looked at the variation in 5 and 10 years survival, and probability of death from CML based on different age groups in the US adult populations from 2000-2019 via SEER.gov database. Methods: We used Incidence -Based Mortality SEER Research Data, 17 Registries, Nov 2021 Sub (2000-2019), which provides the broadest geographic coverage for incidence-based mortality rates and is roughly 26.5% of the US population (based on the 2020 Census) for the calculation of Incidence-Based Mortality and Survival rates of CML. SEER*Stat 8.4.1 was used to obtain cause-specific survival, with Death due to cancer. We used Join-point software, version 5.0.2. by the National Cancer Institute, annually to create log-linear time trends. Results: Fig 1: CML 10-Year Survival rates per 100,000 and age-adjusted to the 2000 US standard population by age groups (50-79) Our results revealed a significant change in the probability of dying from CML for age groups 60-64 and 70-74. The Average Annual percent Change (AAPC) for incidence Based Mortality by age groups were 3.71%*(95% CI 1.55 to 6.42 ), 2.87%*(95% CI 0.74 to 5.56), 6.09%*(95% CI 2.97 to 9.03 ), 2.33%*(95% CI 0.38 to 5.84), 4.86%*(95% CI 2.74 to 7.18), 3.38%* (95% CI 2.08 to 4.97) for age groups 50-54, 55-59,60-64,65-69,70-74, and 75-79 respectively. The trend for 10 year survival shows increasing survival across all age groups. The AAPC for 10 year survival were 1.74%*(95% CI 0.08 to 4.04), 4.08%*(95% CI 2.25 to 7.33), 2.52% (95% CI -0.22 to 6.21), 5.29%* (95% CI 3.34 to 7.70), 5.42%* (95% CI 2.13 to 9.71), 2.29% (95% CI 1.88 to 6.92). Conclusion: Our Study shows a significant difference in the survival rates at 10 years based on age groups, with a pattern of lower survival rates and higher probability of death from CML with increased age at diagnosis.

The Impact of Delayed Venetoclax Initiation on Overall Survival in Acute Myeloid Leukemia

Introduction The use of venetoclax in acute myeloid leukemia (AML) has become routine - following FDA approval, approximately half of patients with AML have received venetoclax. However, the outcomes of the timing of venetoclax initiation remain unknown. Such outcomes are of notable importance, as it is often critical to allow time for patient optimization and to await molecular profiling - particularly as ongoing research contributes to a deeper understanding of the mechanisms of venetoclax sensitivity and resistance. A critical real-world limitation is that the turnaround times of complete molecular profiling via NGS assays are frequently 14 to 21 days. Therefore, we performed the first stratified analysis that details the impact of delays in venetoclax-based regimen initiation on overall survival. Methods We analyzed 185 patients with AML treated with venetoclax and a hypomethylating agent (azacitidine or decitabine; VEN+HMA) from January 1, 2018 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We recorded baseline patient-related and disease characteristics, including age, ECOG, Charlson comorbidity index (CCI) scores, molecular profiling and ELN 2022 cytogenetic risk, dates of regimen initiation, and survival. We individually analyzed the first-line and relapsed or refractory (RR) groups. In each group, we separated patients into three cohorts: those that started therapy in 0-7 days, 8-14 days, and 15 days and beyond. We used the D'Agostino & Pearson method for normality testing and the Mann-Whitney test for between-group comparisons. We applied the Bonferroni correction if multiple comparisons were made. We analyzed survival by the Kaplan-Meier method, with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. Results Of the 185 patients treated with venetoclax and a hypomethylating agent, 100 (54.1%) were in the first-line setting, and 85 (45.9%) had RR AML. In the first-line setting, the median time to initiation of VEN+HMA was 5 days (range: 0-69 days). Compared with the first-line setting, VEN+HMA for RR AML was started at a significantly longer time from the date of disease progression at a median of 11.5 days (range: 0-110 days, p < 0.0001). In the first-line setting, the median OS was significantly worse for earlier initiation of VEN+HMA at 5.8 months for the 0-7 day cohort, compared with 8.9 months for the 8-14 day cohort and 12.7 months for 15 days and beyond (p = 0.023, Figure A). Similarly, in RR AML, the median OS was also significantly worse in the 0-7 day cohort at 4.9 months - but significantly better in the 8-14 day cohort at 10.5 months compared with the 15-day and beyond cohort at 7.1 months (p = 0.027, Figure B). Discussion In the first-line setting, short delays in initiating VEN+HMA do not adversely impact survival. This approach may be preferred to optimize comorbidities and disease-related complications while awaiting the results of molecular profiling. In RR AML, delays in therapy initiation also do not appear to decrease survival - although the optimal therapeutic window appears to be shorter. Taken together, these findings are the first to suggest that with lower-intensity venetoclax-based strategies, short delays in treatment for patient optimization and comprehensive molecular profiling are associated with improved survival.

Impact of COVID-19 Pandemic on Overall Survival in Acute Myeloid Leukemia: A Comprehensive Cancer Center Experience of 627 Patients

Background: The COVID-19 pandemic impacted cancer care nationwide. Challenges with frequent clinic visits for lab draws, administration of multi-agent chemotherapy regimens, and scarcity of supportive blood products disrupted effective health care delivery. The magnitude of these factors on the survival of patients diagnosed with acute myeloid leukemia (AML) during the pandemic is incompletely described. Methods: We included 627 patients aged ≥18 years with AML treated at VCU Massey Comprehensive Cancer Center from January 1, 2013 to December 31, 2022; we recorded baseline demographics, molecular profiles, induction regimens, and survival data. We defined the pandemic era as March 7, 2020 (coinciding with the first confirmed case in our region) to July 1, 2021 (routine and widespread vaccination available). Survival analyses were performed using the Kaplan-Meier method and compared using the log-rank test. ECOG and Charlson comorbidity index (CCI) scores were compared using the Mann-Whitney test; we used the D'Agostino & Pearson method for normality testing. Results: The median age of patients who received intensive chemotherapy (7+3, CPX-351, FLAG-IDA, MEC, or HAM) in the COVID-19 pandemic cohort was 60 years (range, 28.5-69.6) vs 58 years (range, 19.6-81.4) for the non-COVID-19 cohort (p = 0.932). The median ECOG performance status in both cohorts was 1 (range, 0-4, p = 0.658). The median Charlson comorbidity index (CCI) score for patients for both groups was 4 (range, 0-9 vs 2-9, p = 0.909). Patients who were initiated on lower-intensity therapies (e.g., a hypomethylating agent with or without venetoclax) during the COVID-19 pandemic had a median age of 75 (range, 61.9-85.1) years vs 73 (range, 26.1-92.0) years in the non-COVID-19 cohort (p=0.438). The median ECOG performance status for the COVID-19 cohort was 2 (range, 0-3) vs 1 (range, 0-4) for the non-COVID-19 cohort (p=0.005). CCI score for both groups was 6 (range, 4-12 vs 2-14, p=0.789). We then compared the mOS between the COVID-19 and non-COVID-19 eras for both intensive and non-intensive treatment cohorts. For patients treated with intensive chemotherapy during the pandemic, the mOS was 25.1 months in the COVID-19 cohort vs 18.0 months (p = 0.173) in the non-COVID-19 cohort. For patients treated with non-intensive regimens, mOS was 6.6 months in the COVID-19 cohort vs 6.0 months in the non-COVID-19 cohort (p = 0.963). Patients with relapsed disease who were retreated with intensive chemotherapy during the COVID-19 pandemic had a mOS of 6.5 months compared to 8.2 months (p = 0.094) in the non-COVID-19 cohort. Relapsed patients in the lower-intensity COVID-19 cohort had a significantly shorter mOS of 1.8 months compared to 5.2 months in the non-COVID-19 cohort (p = 0.005). Regardless of therapy, the mOS was shorter in the COVID-19 cohort at 11.0 months compared to 13.4 months in the non-COVID-19 cohort (p = 0.191). Relapsed or refractory patients in the COVID-19 cohort had significantly shorter mOS at 5.8 months compared to 8.0 months in the non-COVID-19 cohort (p = 0.019). Discussion: The median overall survival appears shorter for patients diagnosed with AML during the COVID-19 pandemic. Patients with relapsed or refractory AML appeared to have significantly worse overall survival during the COVID-19 pandemic, particularly with lower-intensity therapies. Patients treated with less-intensive regimens are generally older with increased frailty, raising concern for lack of physiologic reserve. These results convey a lesson in preparation for future healthcare crises.

The CNS Microenvironment Promotes Leukemia Cell Survival By Disrupting Tumour Suppression and Cell Cycle Regulation in Pediatric T-Cell Acute Lymphoblastic Leukemia

Although modern treatment has improved the overall survival of pediatric T-cell acute lymphoblastic leukemia (T-ALL) greatly, more than 20% of all children diagnosed with T-ALL relapse (Fattizzo et al., 2020). Approximately 30% of all relapse cases occur in the central nervous system (CNS) and CNS involvement is considered a major risk factor for treatment failure and increased mortality (Frishman-Levy & Izraeli, 2017). To date, CNS directed therapy is considered essential for complete cure to prevent relapse in the CNS, leading to severe neurotoxic effects and long-term adverse complications (Thastrup et al., 2022). Due to lack of knowledge regarding the mechanisms promoting leukemia survival in the CNS and absence of reliable biomarkers to predict CNS involvement, the development of new therapeutic drugs able to eradicate leukemia cells in the CNS is not progressing, therefor limiting the overall survival of pediatric T-ALL patients (Thastrup et al., 2022). To identify genetic alterations in CNS involved T-ALL we performed an RNA-sequencing analysis coupled with a gene set enrichment analysis (GSEA) studying pediatric T-ALL patient samples obtained from the TARGET initiative. We included 30 samples from patients at initial diagnosis subdivided by degree of CNS involvement, ranging from CNS1 (no CNS disease) to CNS3 (advanced CNS disease), as well as 13 samples from patients who had relapsed in the CNS. Our analysis identified distinct genetic alterations in CNS involved T-ALL and CNS relapse compared to diagnosis samples without CNS implications, with dysregulation of genes involved in tumour suppression, apoptosis and self-renewal. CNS relapsed patients were distinguished from diagnosis patients by a distinct gene expression pattern, with an upregulation of many genes, including cell cycle inhibitor CDKN2A and transcription factor HOXA3 (Figure 1A). We also found disruption of many signaling pathways, including enrichment of the p53 pathway in patients who relapsed in the CNS compared to non-CNS involved diagnosis patients. To further study the effects of CNS implications in pediatric T-ALL, we established a co-culture of pediatric T-ALL cell lines (n=4) and a glioblastoma cell line (U87-MG). Following co-culture, changes to mRNA expression was evaluated by RT-qPCR, revealing dysregulation of many genes, including an upregulation of cancer stem cell marker and cell adhesion molecule CD44 (Chen et al., 2018). Moreover, T-ALL cells grown in CNS co-culture displayed a downregulation of tumour suppressor TP53 and an upregulation of its negative regulator, proto-oncogene MDM2 when compared to cells grown in suspension (Figure 1B) (Hernandez Borrero & El-Deiry, 2021). Co-culture also induced pro-survival isoform switching of BCL2L1 (BCLX), part of the BCL family of apoptotic regulators (Daniel et al., 2013). Together, these results suggest an increased survival and loss of tumour suppression of T-ALL cells when grown in direct contact with CNS derived cells. To determine effects of the CNS microenvironment on therapy resistance, T-ALL cell lines grown in co-culture were exposed to 2µM methotrexate, a chemotherapeutic agent used during CNS directed therapy (Thastrup et al., 2022). Leukemia cells in co-culture displayed an increased survival compared to cells grown in suspension following 48 hours of methotrexate exposure, as well as dysregulation of several genes involved in cell survival, self-renewal and cell cycle regulation. Cyclins and CDKs involved in controlling cell cycle progression were differentially expressed in co-culture versus suspension, with a downregulation of CDK2 and cyclin A, crucial for S/G2 transition, as well as CDK1 and cyclin B, responsible for G2/M transition (Leal-Esteban & Fajas, 2020). These results indicate a shift towards cell cycle arrest and senescence of T-ALL cells during methotrexate exposure in CNS co-culture, potentially promoting leukemia survival and chemoresistance. Taken together, our findings indicate a differential gene expression pattern in CNS involved T-ALL, which could potentially be utilized as predicative biomarkers to properly identify patients at risk of CNS relapse. CNS co-culture revealed cell cycle alterations and loss of proliferation markers, promoting senescence and therapy resistance, which could serve as potential therapeutic targets.

Impact of Racial, Age, Marital, and Socioeconomic Status Disparities on the Survival of Hairy Cell Leukemia: A Population-Based Study

Introduction Several studies report excellent long-term overall survival (OS) for patients with hairy cell leukemia (HCL). Other studies show that racial and socioeconomic disparities impact outcomes among cancer patients. In the US, healthcare access is closely related to insurance coverage. Hispanics are the least likely to have insurance of any racial or ethnic group; they have lower levels of educational attainment and had a poverty rate of 17% in 2019-2020. Age and Marital status were also demonstrated to be independent prognostic factors in other cancers. We assessed the effect of race, age, marital and socioeconomic status on the overall survival and cause-specific mortality of HCL, which is generally associated with a good outcome. Methods We queried the Surveillance, Epidemiology, and End Results (SEER) registry for patients diagnosed with HCL between 2000 and 2020. Data were collected on age at diagnosis, race/ethnicity, marital status, socioeconomic status as defined by the annual household income data, time and cause of death. The chi-squared test was used to assess the association between two categorical variables, the Mann-Whitney U test and the Kruskal-Wallis test were used for continuous data that were non-normally distributed. The Kaplan-Meier method was used for survival analysis. Statistically significant differences between the curves were estimated using the 2 tailed log-rank test. Cox proportional hazard models were used for multivariable analysis. Results We identified 5299 HCL patients, survival data was available for 5280 patients. The median age was 59; interquartile range (IQR) (49-71) In the study population, 38% (2019) patients were ≥ 65 years at diagnosis, and 61% (3259) were married. Male patients (4188) represented 79% of HCL. Hispanic patients (491) represented 9% of patients. Eight percent (427) had an annual household income of <$50K. The median OS for the study population was 84 months; IQR (32-148.8). The HCL case fatality rate was 11% for the entire study population. Hispanic ethnicity was associated with reduced OS with a median OS of 70 months; IQR (23.5-134.5) compared to 86 months; IQR (32-150) for the rest of the population (p ≤0.001). Patients diagnosed at age <65 years old had a higher median OS of 105 months; IQR (47-170) vs 55 months; IQR (20-106) in patients ≥65 years old (p ≤0.001). Married patients had a better median OS of 89 months; IQR (36-156) compared to 69 months; IQR (24-134) in the unmarried group (p <0.001). Patients with an annual household income ≥$50K had a better median OS of 85 months; IQR (32-150) than patients with a lower household annual income of 76 months; IQR (27-136) (p=0.012). HCL cause-specific mortality was significantly better in the patients diagnosed at age <65 (5%), married group (10%) and household annual income ≥$50K (11%) compared to patients diagnosed ≥65 (22%), unmarried (15%) and patients with household annual income <$50K (16%) (p ≤0.001, ≤0.001, ≤0.001 respectively), but was not significant based on race, Hispanic (10%) versus Non-Hispanic ethnicity (11%) (p =0.492). Conclusions Our analysis shows a significantly better effect of age <65, marital status being married, and socioeconomic status with household annual income ≥$50K on the overall survival and cause-specific mortality of patients with HCL. Patients of Hispanic ethnicity had reduced overall survival but no difference in HCL cause-specific mortality. Hispanic, older, unmarried, and patients with low annual income may need more support, and resources to improve their outcomes and eliminate disparities.

Unraveling Clonal Evolution and Mechanisms of Treatment Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal FLT3 N676K

Our understanding of how individual mutations, whether present in all or just a subset of the leukemia cells, affect cellular response to therapy is limited. Herein, we investigate the impact of different therapies on survival, evolution, and resistance patterns in an acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3 and subclonal FLT3 N676K. Leukemic bone marrow expressing KMT2A::MLLT3-mCherry in all cells and FLT3 N676K-GFP in 40% of cells was re-transplanted into sublethally irradiated recipients. Upon engraftment, recipients were subjected to different treatments, including chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was administered for 28 days, while the control group received vehicle ( Fig 1a). Each treatment prolonged survival with a median latency of 30 days for chemotherapy 37.5 days for AC220, 42 days for chemotherapy+AC220, and 45 days for AC220+Trametenib, and 25.5 days for control mice. At sacrifice, all mice displayed splenomegaly and leukocytosis. The treatment impacted the evolution of the KMT2A:: MLLT3+ FLT3 N676K cells and they expanded to clonal dominance in control and chemotherapy-treated mice. However, three distinct patterns of evolution were detected in AC220-treated mice with the FLT3 N676K-containing cells either expanding to clonal dominance (>80% of KMT2A:: MLLT3+ FLT3 N676K), the FLT3 N676K-containing cells being eradicated (>80% of KMT2A::MLLT3-alone), or dual similar sized clones expressing KMT2A:: MLLT3-alone or KMT2A::MLLT3+ FLT3 N676K(Fig1 b).Eradication of the FLT3 N676K-cells was rare, predominantly observed upon AC220+Trametinib treatment and the frequency of dual clones increased when chemotherapy was followed by AC220, in line with treatment affecting the evolution of genetically distinct clones. RNA-sequencing was used to determine altered gene expression patterns upon resistance and unveiled three major clusters including 1) mice expressing KMT2A:: MLLT3-alone, 2) control and chemotherapy-treated mice, and 3) AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting potential resistance mechanisms through activation of the MEK/ERK-pathway. All leukemias had a myeloid Cd11b + phenotype. However, the control and chemotherapy-treated leukemias also displayed an aberrant expression of the lymphoid marker B220 and showed enrichment of gene sets correlating with stemness and oxidative phosphorylation, that suggested a switch in cellular phenotype and metabolic state. In contrast, AC220-treated leukemias lacked or had a low fraction of B220 + cells and showed enrichment of gene sets correlating with granulocyte-monocyte progenitors (GMP) and immune regulatory pathways (Fig 1 c). Acquired AC220-resistance mutations was rare (n=2), suggesting activation of alternative pathways upon resistance. Re-treatment of established cell lines from treated animals with AC220 showed that the cells were resistant. To uncover mechanisms of resistance at the cellular level, single cell RNA-sequencing was performed on four mice and revealed the emergence of distinct cellular subpopulations in AC220-treated mice, with a strong GMP signature and upregulation of the homeobox gene Six1(Fig 1d).Overexpression of Six1 in all AC220-treated leukemias was further confirmed by bulk RNA-sequencing. Interestingly, Six1 has previously been described as a regulator for leukemia stem cell maintenance in AML and under Wnt/ß-catenin signalling control. Subsequent drug assays showed AC220-treated cells exhibited sensitivity to Six1 pharmacological inhibition, suggesting a dependency on Six1, as well as sensitivity to Wnt/ß-catenin inhibition. Taken together, the treatment given affected survival and impacted evolution of the FLT3 N676K-leukemia cells. The general absence of acquired mutations in most mice suggests that target-independent mechanisms may underline acquired resistance in most mice, and we propose the Six1/Wnt/ß-catenin axis as a potential vulnerability upon AC220-resistance.

Phase 2 Study of CPX-351 in Combination with Venetoclax in Patients with Newly Diagnosed, High Risk Acute Myeloid Leukemia

Background CPX-351 is a nanoscale liposome of a fixed 5:1 molar ratio of cytarabine and daunorubicin which has improved survival of secondary acute myeloid leukemia (AML) 1. Venetoclax (ven) is a BCL2 inhibitor which, in combination with azacitidine or cytarabine, has become the standard of care for elderly AML. Ven has shown synergism with chemotherapy, with high complete remission (CR) rates in newly diagnosed (ND) and relapsed / refractory (RR) AML 2. In this study, we aim to assess the efficacy and safety of CPX-351 and ven (CPX-ven) in RR and ND AML. Methods This was a phase 2 study with a lead-in safety phase. Enrolment was allowed if the blasts were ≥10%. Patients (pts) age ≥18 with RR AML were eligible for the safety lead-in phase (LIP) or arm A. Arm B enrolled pts age 18 - 69 with ND AML. Other inclusion criteria included adequate liver, renal and cardiac function, and ECOG performance status ≤2. Pts with CNS involvement or prior CPX-351 were excluded. The recommend phase 2 dose was CPX-351 (daunorubicin 44 mg/m 2 and cytarabine 100 mg/m 2 IV on D1, 3 and 5 of induction, and daunorubicin 22 mg/m2 IV on D1 and 3 during consolidation) combined with ven on D2-8 of each cycle. Ven was dosed at 300mg daily and reduced to 150mg or 50mg daily for pts on a moderate or strong CYP3A inhibitors, respectively. The primary outcome measure was complete remission (CR) or CR with incomplete count recovery (CRi). Here, we report the outcomes of arm B including pts with ND AML. Results 14 pts were enrolled on arm B. One pt withdrew and one was too early in treatment, therefore 12 were evaluable. Baseline characteristics are shown in Table 1. The median age was 59.5 years (range, 44 - 69); Four (33.3%) were female. All 12 (100%) pts were classified as adverse risk per the 2017 European Leukemia Net Guidelines. Two (16.7%) pts had treatment for a prior non-myeloid malignancy (therapy-related AML). Eight (66.7%) had an antecedent haematological disorder (secondary AML), of whom seven had treatment (median 1 line, range 1 - 2). Four (33.3%) pts had a complex karyotype, of whom three harbored a TP53 mutation. Nine (75.0%) pts achieved a response, including four CR (33.3%), five CRi (41.6%), and one induction mortality (8.3%) prior to disease assessment. 3/4 (75.0%) pts with complex cytogenetics (including 2/3 (66.7%) with a TP53 mutation) achieved a CR/CRi. Of the secondary AML pts with an evaluable response, 5/7 (71.4%) achieved a CR/CRi. Of the nine pts achieving CR/CRi, five (55.5%) were negative for minimal residual disease (MRD) by flow cytometry. The median number of cycles to CR/CRi was one (range 1 - 2). The median overall survival (OS) was 12.9 months (95% CI, 3.1 - not reached, NR, Figure 1). The median relapse-free survival (RFS) was 8.7 months (95% CI, 3.1 -NR). 7/9 (77.8%) of responding pts underwent an allogeneic stem cell transplant (SCT) after attaining CR/CRi. There were 90 adverse events (AE) in 10 (83.3%) pts. 80 (88.9%) were grade 1 or 2, 10 (11.1%) were grade ≥3. The most common AE was gastrointestinal with seven (7.8%) occurrences. Four (33.3%) pts developed grade ≥3 infections; of these, one (8.3%) died. The cause of death was neutropenic septic shock due to pneumonia. Conclusion CPX-ven is a feasible option for remission induction in pts with high-risk, newly diagnosed AML. The combination produced high rates of response and MRD negativity in a cohort of pts with adverse risk features, including pts with adverse karyotype and AML following prior HMA-based treatment, allowing them to proceed with SCT. Toxicities are as expected for an intensive chemotherapy regimen. Further studies are needed to confirm the efficacy of this combination.

Mitochondrial STAT3 Plays a Critical Role in Survival of AML Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be critical to the survival of acute myeloid leukemia (AML) cells. It has been previously shown to mediate oxidative phosphorylation (OXPHOS) in acute myeloid leukemia bulk and stem cells via its transcriptional regulation of the amino acid transporter SLC1A5 and thereby glutamine uptake to AML cells (Amaya et al. Blood 2022). Further, STAT3 has been shown to regulate the transcription of important mitochondrial genes such as MCL-1 (Shastri et al. JCI 2018). Although STAT3 has been previously shown to also translocate to the mitochondria of myeloid malignant cells, its role in the mitochondria is less well defined in this cell population. In this study, we sought to understand the role of mitochondrial STAT3 in AML bulk leukemia and leukemia stem cell population. Methods: Human primary AML samples and MOLM-13 cells were utilized for experiments. To assess STAT3 localization to the mitochondria, mitochondrial fractions were isolated followed by Western Blot analysis. Flow cytometry-based imaging (ImageStream) was also utilized to quantify localization of STAT3 to the mitochondria. Mitochondrial mass was assessed using flow cytometry based MitoTracker assays. Metabolomic assays were performed by mass spectrometry at the University of Colorado metabolomics core. Viability assays were performed with flow cytometry using Annexin V and Dapi stains. Patient derived xenograft models were performed as previously described (Amaya et al. Blood 2022) in NSG-S mouse with Busulfan conditioning. Results: In this study, we show that primary human AML cells have constitutive STAT3 activation via phosphorylation of S727, which has been shown to signal mitochondrial localization. Further, we show that STAT3 is localized in the mitochondria of leukemia cells in primary patient samples and the cell line MOLM-13. Using genetic (siRNA) and pharmacologic (Stattic) inhibition of STAT3, we show a decrease in pSTAT3 at S727, with subsequent decreased translocation of STAT3 to the mitochondria. This leads to mitochondrial dysfunction including decreased TCA cycle metabolites, as well as decreased intracellular ATP upon STAT3 inhibition. STAT3 inhibition also leads to decreased mitochondrial mass suggesting autophagy upon mitochondrial STAT3 (mitoSTAT3) inhibition. To assess whether mitoSTAT3 is functionally important, we performed viability assays in MOLM-13, as well as leukemia stem cells isolated from primary AML cells. Inhibition of mitochondrial STAT3 leads to decreased viability in MOLM-13 and primary AML cells, and reduced engraftment in PDX mouse models using primary AML samples (Figure 1A), suggesting a decrease in leukemia stem cell potential. Interestingly, STAT3 inhibition with Stattic is able to overcome venetoclax resistance for primary AML patient samples in vitro (Figure 1B). Conclusions: In this study, we show mitoSTAT3 plays a key role in mitochondrial function of AML cells. mitoSTAT3 inhibition leads to mitochondrial dysfunction, a decrease in mitochondrial mass and eventually cell death. We show that Stattic is an effective inhibitor of mitoSTAT3 in AML, leading to cell death and decreased leukemia stem cell function. Further studies are needed to assess the mechanistic function of STAT3 in the mitochondria.

Impact of Facility Type on Survival in Chronic Myelomonocytic Leukemia: A Propensity Score Matched, National Cancer Database Analysis

Introduction Chronic myelomonocytic leukemia (CMML) is an MDS/MPN overlap syndrome with a heterogenous clinical presentation and limited therapeutic options. Given its rarity, we hypothesize that there may be disparate outcomes based on the type of treatment facility (academic versus community) similar to what has been observed in other myeloid malignancies such as acute myeloid leukemia (VR Bhatt AJH 2017). To that end, we performed an analysis of the National Cancer Database (NCDB) to assess the impact of academic status of the facility on overall survival (OS) in patients with CMML. Methods We identified patients in the NCDB by the ICD-O-3 code for CMML (9945); we excluded patients who did not receive treatment and those who received all or part of their treatment outside of the reporting facility. Importantly, baseline clinical characteristics, laboratory/mutational data, and treatment details are generally not available in the NCDB. Academic centers were defined as those with a peer-reviewed NCI-designated cancer center support grant or university medical school that provide postgraduate medical education in at least four program areas, as defined by the Commission on Cancer Accreditation program. The primary outcome was OS. Propensity score matching (PSM) was performed and after backwards selection, the following variables were used for matching: age, race, median income, urban/rural, education, Charlson comorbidity index (CCI). Cox proportional hazard multivariable regression was employed to identify independent predictors of survival in the PSM population. Results A total of 6290 patients were identified. Baseline characteristics based on facility type are shown in Table 1. Patients treated at an academic center were younger, with a higher prevalence of Black race and lower comorbidity burden. Academic centers also had a patient population with a significantly higher median household income, more private insurance, and higher education level as compared to non-academic centers. The median follow-up time was 14.6 months (95% CI 0.4-89.7). The median OS for the entire cohort was 59 months (95% CI 56.7-62.0) and the median survival was significantly longer in patients treated at an academic center (60.6 months [95% CI 57.5-64.8]) as compared to those treated at a non-academic center (57.3 months [95% CI 53.4-61.8]). In the PSM analysis, a cohort of 1884 patients treated at an academic center were matched to 1895 patients treated at a non-academic center. In an unadjusted analysis, there was no significant difference in median OS (p=0.3) between the academic and non-academic groups. However, in a multivariable analysis of the PSM populations that accounted for age, race, CCI, insurance, distance to center, and median income, patients treated at an academic center had a significantly improved survival as compared to those treated at a non-academic center (HR 0.82, 95% CI 0.71-0.94, p = 0.005, Figure 1). Discussion We found significant differences in the characteristics of CMML patients treated at academic versus non-academic centers. After PSM and multivariable adjustment, survival was significantly improved with treatment at an academic center versus non-academic. Although limited in the depth of information about CMML prognostic features and treatments, this relatively large analysis of CMML patients suggests that management of this rare disease at an academic center may be associated with improved survival outcomes. Future prospective studies to ascertain the underlying causes for survival discrepancy in CMML at academic versus non-academic centers may help identify patients that should be triaged to an academic facility.

Thrombosis and Bleeding Outcomes Among Veterans with Chronic Myeloid Leukemia and Association with Agent Orange Exposure

The incidence of chronic myeloid leukemia (CML) in the United States has been reported to be 1.75/100,000. Older age and male patients are more likely to be diagnosed with CML (Chen Y et al. Trends in chronic myeloid leukemia incidence and survival in the United States from 1975 to 2009. Leuk Lymphoma. 2013 Jul;54(7):1411-7). Since their introduction in 2000, the mainstay of treatment for CML has been tyrosine kinase inhibitors (TKIs). These medications are generally well-tolerated. Of the TKIs in use today, Ponatinib is known to have adverse vascular events but is not recommended for use in the first line. Nilotinib has also been shown to have adverse arterial events, which tend to occur late in the treatment course (Rea D. Management of adverse events associated with tyrosine kinase inhibitors in chronic myeloid leukemia. Ann Hematol. 2015 Apr;94 Suppl 2:S149-58.). In this study, we evaluated the thrombosis and bleeding complications of CML in the era of TKIs. The Sergeant First Class (SFC) Heath Robinson Honoring our Promise to Address Comprehensive Toxics (PACT) Act is a law that expanded Veterans' health care and benefits to those with exposure to carcinogens such as Agent Orange (AO). The PACT Act has a list of presumptive conditions due to exposure to AO. Myeloid malignancies have not been added to the presumptive list due to insufficient evidence from the Veterans and Agent Orange: Update 11 (2018). We also investigated the association of CML and AO exposure. We performed a case control study from January 1, 2006 - January 26, 2023, by utilizing the Veterans Affairs Informatics and Computing Infrastructure (VINCI) database to characterize the association of AO exposure with development of CML. ICD-9 and -10 codes identified Veterans with CML, arterial thrombosis (AT), venous thrombosis (VT), and bleeding. Age-, sex-, and race-matched controls (1:1) were selected from VINCI, who are Veterans without CML from Illinois, the state most representative of the U.S. population. Exposure to Agent Orange was self-reported and verified through Veterans' duration and location of service. We stratified the Veterans according to birth year (born before or after 1960) as those born before 1960 are most likely to have AO exposure. Qualitative data were compared by chi-square tests. When comparing all Veterans with CML and Veterans without CML, AO exposure is associated with the development of CML, OR 1.71, 95% CI 1.63-1.80, p<0.0001 (Figure 1). When adjusting for birth year and comparing all Veterans with CML unmatched controls from Illinois, there remains a statistically significant association, OR 1.21, 95% CI 1.14-1.28, p<0.0001. Further comparison to a group of matched controls is also statistically significant, OR 1.14, 95% CI 1.06-1.24, p=0.0007. Veterans born before 1960 and Males were significantly associated with development of CML, OR 6.25 95% CI 5.89-6.63 and OR 4.79 95% CI 4.29-5.36, both p<0.0001. Table 1 details the demographics and clinical characteristics among Veterans with or without CML, with or without AO exposure, and matched and unmatched controls. When comparing all Veterans with CML and matched controls, we observed an association of CML with bleeding (OR 3.95, 95% CI 3.70-4.22), AT (OR 2.58, 95% CI 2.43-2.74), and VT (OR 2.19, 95% CI 1.98-2.41), all p<0.0001. We then investigated whether AO exposure increases the risk of AT, VT, and among Veterans with CML. When adjusting for birth year, we observed an increased risk of AT (OR 1.35, 95% CI 1.21-1.51, p<0.0001) and bleeding (OR 1.13, 95% CI 1.02-1.26, p=0.02) among Veterans with CML with AO exposure but not VT (OR 1.17, 95% CI 0.99-1.37, p=0.05). Our analysis demonstrates a positive association between previous exposure to AO and subsequent development of CML in Veterans. These findings suggest that CML is the second myeloid malignancy to be associated with AO exposure apart from myeloproliferative neoplasms (J Clin Oncology 2023 41:16_suppl, 7011). We also demonstrate a strong association in patients with CML and outcomes including AT, VT, and bleeding. This is the first large cohort study to demonstrate these associations. These outcomes may be affected by tyrosine kinase inhibitors for the treatment of CML. Further investigation will include overall survival data, specific prognostic factors related to CML, cardiovascular risk factors, and information on the timing of AT, VT, and bleeding in relation to disease diagnosis and treatment onset.

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

Epitope Edited Hematopoietic Stem Cells to Enable Synergistic Immunotherapy Combinations for Acute Myeloid Leukemia

Despite treatment advances, acute myeloid leukemia (AML) is still associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CAR-T cells, bispecific and toxin conjugated antibodies (mAb), demonstrated clinical efficacy when targeting dispensable lineage antigens (Ag), such as CD19 in B-ALL, the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. Suitable targets are shared with healthy progenitor or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. Several AML immunotherapies are currently under development, but their use is restricted to a limited time window, likely insufficient for disease eradication. To address these issues, we reasoned that precise modification of the targeted epitopes in donor HSPC used in HSCT results in loss of mAb recognition, without affecting normal protein expression, regulation, and function ( Nature, accepted). Epitope editing allows targeting genes essential for leukemia survival regardless of shared expression or functional role in normal HSPC, thus minimizing the risk of tumor immune escape by Ag loss or downregulation. Cytokine receptors such as FLT3, KIT and CD123 are found in >85% of AML cases and their mutation (e.g., FLT3-ITD) or overexpression is associated with poor prognosis. We identified amino acid substitutions in the FLT3, KIT and CD123 extracellular domains that preserve physiologic surface expression, ligand-binding, kinase phosphorylation, colony-forming capacity, proliferative response, transcriptional and phospho-proteomic profile of CD34+ HSPCs but avoid detection by a therapeutic monoclonal Ab. Cells expressing these variants were resistant to CAR-T killing and did not induce CAR activation and proliferation during in vitro co-culture. We were able to introduce these mutations into CD34+ HSPCs by adenine base editing (ABE) with high efficiencies (90%, 85% and 75% for FLT3, KIT and CD123, respectively) without the need for dsDNA breaks. After xenotransplant into NBSGW mice, FLT3, KIT or CD123 epitope-editedHSPC sustained long-term multilineage hematopoiesis indicating editing and preserved functionality of HSCs. Upon treatment with FLT3 CAR-T in vivo, we observed sparing of human CD34+38- HSPCs, granulo-mono progenitors (GMP), B-cells and B-progenitors in the bone marrow of mice engrafted with FLT3-edited HSPCs compared to AAVS1 controls. Treatment with CD123 CAR-T cells showed protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. We next generated advanced in vivo models with co-engraftment of human HSPCs and patient-derived AML xenografts (PDX), to demonstrate the selective resistance of epitope edited HSPCs and their progeny, while PDX were eradicated by FLT3 or CD123 CAR-T. To further enhance therapeutic efficacy, we are now exploring the combination of epitope-edited HSPCs and pharmacological agents with potential synergistic effects with CAR-T cells. FLT3 tyrosine kinase inhibitors (e.g., Crenolanib) have the potential to enforce surface expression of FLT3 by impairing its intracellular recycling, thus enhancing CAR-T mediated killing, but their use may be limited by overlapping toxicities on healthy hematopoiesis, in particular in the post-HSCT setting. To assess if epitope-editing could prevent not only CAR- but also TKI-mediated toxicities, we treated mice co-engrafted with FLT3- or AAVS1- edited HSPCs and a FLT3-ITD+ AML PDX with combinations of FLT3-CAR and a 2-week course of Crenolanib. Leukemia burden was reduced by Crenolanib alone and eradicated by FLT3-CAR-T. Critically, we observed preservation of healthy hematopoietic lineages only in mice engrafted with epitope-edited HSPCs, including the CAR+Crenolanib group, while AAVS1 controls showed overlapping off-tumor toxicity when treated with the CAR+Crenolanib combination. In conclusion, we believe that epitope edited HSPCs may not only enable safe and effective CAR-T immunotherapies for AML, but also allow their combination with pharmacological blockade of leukemia survival/proliferative pathways to achieve synthetic lethality mechanisms, while still avoiding dose-limiting toxicities. Further exploration of immunotherapy-synergistic combinations will be fundamental to improve the outcomes of difficult-to-treat high-risk AML patients.

Sustained MYB Activity Is Necessary for Oncogenic Transcription in KMT2A-Rearranged Acute Lymphoblastic Leukemia through Enhancer-Promoter Interactions and Epigenetic Modifications at Enhancers

Aberrant enhancer activity is a hallmark of many cancers including KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive leukemia subtype with an event-free survival of 19-45% in infant-ALL (<1 year of age) and 50-59% in childhood-ALL cases. Targeting enhancer activity may provide a novel therapeutic strategy for this hard-to-treat disease. Transcription factors are key effectors of enhancer activity. They recognise and bind to specific DNA sequences at enhancers, driving the expression of target genes from these distal loci. MYB is an important hematopoietic transcription factor that is commonly overexpressed in KMT2A-rearranged leukemias and is required for leukemia survival, where loss of MYB has been shown to abrogate the leukemia phenotype. However, the precise mechanism by which MYB contributes to enhancer function, and the stages of enhancer activation at which it acts are unknown. We employed a well-characterized chromatin anchoring system to interrogate MYB function and determine whether it can recruit specific enhancer activities de novo. By fusing the MYB transactivation domain (MYB TA) to the Tet repressor (TetR) DNA binding domain, MYB TA is directed to an array of Tet operator ( TetO) sequences incorporated into a neutral genomic region devoid of native gene regulatory elements. We found that MYB TA alone is sufficient to recruit key enhancer-associated proteins, including P300, Brd4 and Mediator to the TetO locus. We observed MYB TA-dependent deposition of H3K27ac, increased chromatin accessibility and transcription not just at the TetO locus, but remarkably, also at regions more than 50kb away. Using the chromosome conformation capture (3C) technique NG Capture-C, we observed MYB TA-induced DNA interactions between TetO and the distal sites of transcription, as well as more long-range interactions up to 400kb distal to where MYB TA is bound. Together, these observations suggest that MYB TA binding alone is sufficient to establish an enhancer-like regulatory element, which activates distant cryptic promoters. This activity is dependent on the continued presence of MYB TA, as disruption of MYB TA binding results in a rapid loss of transcription and acquired chromatin features, implicating MYB in enhancer maintenance as well as initiation. To confirm our observations in a disease context, we tested the requirement for MYB at endogenous enhancers in SEM cells, a model of KMT2A-rearranged ALL. By stably introducing a degradation tag (FKBP12 F36V) to the endogenous MYB gene, we were able to rapidly induce protein degradation and observe the immediate effects on chromatin features. Using transient transcriptome sequencing, we noted significant transcriptional downregulation of known MYB target genes, including BCL2, MYC, LMO4 and CDK6. We also observed decreases in enhancer H3K27ac levels and eRNA transcription, most marked at MYB-bound loci. To investigate the requirement for MYB in maintaining physical proximity between enhancer and promoter, we performed the base pair-resolution 3C technique Micro-Capture-C (MCC). MCC revealed highly punctate interactions between enhancer elements and their target promoters. At BCL2 and MYC, MYB degradation results in a reduction in interactions with the promoter at discrete regions within the enhancers. This argues for highly localised MYB-dependent activity at enhancers, driving interaction with and activation of distal target promoters. Overall, our data argue for a role for MYB in establishing enhancers de novo, indicating that MYB overexpression may directly result in onco-enhancer activity at key genes in vivo. At a subset of MYB-bound enhancers, the continued presence of MYB is absolutely required to maintain enhancer activity and oncogene upregulation. Further exploration of associated co-factors that mediate MYB-dependent enhancer activity may identify novel targets for therapeutic exploitation.

Differential vascular endothelial cell toxicity of established and novel BCR-ABL tyrosine kinase inhibitors.

BCR-ABL tyrosine kinase inhibitors (TKIs) have dramatically improved survival in Philadelphia chromosome-positive leukemias. Newer BCR-ABL TKIs provide superior cancer outcomes but with increased risk of acute arterial thrombosis, which further increases in patients with cardiovascular comorbidities and mitigates survival benefits compared to imatinib. Recent studies implicate endothelial cell (EC) damage in this toxicity by unknown mechanisms with few side-by-side comparisons of multiple TKIs and with no available data on endothelial impact of recently approved TKIs or novels TKIs being tested in clinical trials. To characterize BCR-ABL TKI induced EC dysfunction we exposed primary human umbilical vein ECs in 2D and 3D culture to clinically relevant concentrations of seven BCR-ABL TKIs and quantified their impact on EC scratch-wound healing, viability, inflammation, and permeability mechanisms. Dasatinib, ponatinib, and nilotinib, the TKIs associated with thrombosis in patients, all significantly impaired EC wound healing, survival, and proliferation compared to imatinib, but only dasatinib and ponatinib impaired cell migration and only nilotinib enhanced EC necrosis. Dasatinib and ponatinib increased leukocyte adhesion to ECs with upregulation of adhesion molecule expression in ECs (ICAM1, VCAM1, and P-selectin) and leukocytes (PSGL1). Dasatinib increased permeability and impaired cell junctional integrity in human engineered microvessels, consistent with its unique association with pleural effusions. Of the new agents, bafetinib decreased EC viability and increased microvessel permeability while asciminib and radotinib did not impact any EC function tested. In summary, the vasculotoxic TKIs (dasatinib, ponatinib, nilotinib) cause EC toxicity but with mechanistic differences, supporting the potential need for drug-specific vasculoprotective strategies. Asciminib and radotinib do not induce EC toxicity at clinically relevant concentrations suggesting a better safety profile.

CXC chemokine ligand 13 and galectin-9 plasma levels collaboratively provide prediction of disease activity and progression-free survival in chronic lymphocytic leukemia

The clinical outcome of lymphocytic leukemia (CLL) is quite heterogeneous. The purpose of this observational study was to investigate the clinical merit of measuring plasma galectin-9 and CXCL-13 concentrations as predictors of CLL activity, prognosis, and early indicators of therapeutic response. These biomarkers were compared with other prognostic indicators, progression-free survival (PFS), time to first treatment (TTT), and overall survival (OS) over a follow-up period (4 years). First, plasma galectin-9 and CXCL-13 concentrations were analyzed in CLL patients at the time of diagnosis as well as healthy controls. Compared to controls, CLL patients had significantly higher serum levels of CXCL-13 and galectin-9. Second, we observed that CLL patients with high soluble CXCL-13 and galectin-9 levels had advanced clinical stages, poor prognosis, 17p del, short PFS, short TTT, and therapy resistance. The levels of CXCL-13, β2-microglobulin, LDH, CD38%, and high grade of Rai-stage were all strongly correlated with the galectin-9 levels. Soluble CXCL-13 and galectin-9 had very good specificity and sensitivity in detecting CLL disease progression and high-risk patients with the superiority of galectin-9 over CXCL-13. Although the two biomarkers were equal in prediction of TTT and treatment response, the soluble CXCL13 was superior in prediction of OS. High CXCL-13 and galectin-9 plasma levels upon CLL diagnosis are associated with disease activity, progression, advanced clinical stages, short periods of PFS, short TTT, and unfavorable treatment response.

Transient TKI-resistant CD44+pBAD+ blasts undergo intrinsic homeostatic adaptation to promote the survival of acute myeloid leukemia in vitro.

Acute myeloid leukemia (AML) patients have frequent mutations in FMS-like receptor tyrosine kinase 3 (FLT3-mut AML), who respond poorly to salvage chemotherapies and targeted therapies such as tyrosine kinase inhibitors (TKIs). Disease relapse is a common reason of treatment failures in FLT3-mut AML patients, but its intracellular refractory mechanism remains to be discovered. In this study, we designed serial in vitro time-course studies to investigate the biomarkers of TKI-resistant blasts and their survival mechanism. First, we found that a group of transient TKI-resistant blasts were CD44+Phosphorylated-BAD (pBAD)+ and that they could initiate the regrowth of blast clusters in vitro. Notably, TKI-treatments upregulated the compensation pathways to promote PIM2/3-mediated phosphorylation of BAD to initiate the blast survival. Next, we discovered a novel process of intracellular adaptive responses in these transient TKI-resistant blasts, including upregulated JAK/STAT signaling pathways for PIM2/3 expressions and activated SOCS1/SOCS3/PIAS2 inhibitory pathways to down-regulate redundant signal transduction and kinase phosphorylation to regain intracellular homeostasis. Finally, we found that the combination of TKIs with TYK2/STAT4 pathways-driven inhibitors could effectively treat FLT3-mut AML in vitro. In summary, our findings reveal that TKI-treatment can activate a JAK/STAT-PIM2/3 axis-mediated signaling pathways to promote the survival of CD44+pBAD+blasts in vitro. Disrupting these TKIs-activated redundant pathways and blast homeostasis could be a novel therapeutic strategy to treat FLT3-mut AML and prevent disease relapse in vivo.