The multi-CDK inhibitor dinaciclib reverses bromo- and extra-terminal domain (BET) inhibitor resistance in acute myeloid leukemia via inhibition of Wnt/ß-catenin signaling.

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/ß-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of ß-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.

Sex-associated differences in frequencies and prognostic impact of recurrent genetic alterations in adult acute myeloid leukemia (Alliance, AMLCG).

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients' sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3-ITD, DNMT3A, NPM1 and WT1 mutations and less often complex karyotype, ASXL1, SRSF2, U2AF1, RUNX1, or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients' sex in analyses of AML biology and prognostication.

Phase 1 study of selinexor in combination with salvage chemotherapy in Adults with relapsed or refractory Acute myeloid leukemia.

Selinexor, an oral inhibitor of the nuclear transport protein Exportin-1, shows promising single-agent activity in clinical trials of relapsed/refractory (R/R) acute myeloid leukemia (AML) and preclinical synergy with topoisomerase (topo) IIα inhibitors. We conducted a phase 1, dose-escalation study of selinexor with mitoxantrone, etoposide, and cytarabine (MEC) in 23 patients aged < 60 years with R/R AML. Due to dose-limiting hyponatremia in 2 patients on dose level 2 (selinexor 40 mg/m2), the maximum tolerated dose was 30 mg/m2. The most common grade ≥ 3 treatment-related non-hematologic toxicities were febrile neutropenia, catheter-related infections, diarrhea, hyponatremia, and sepsis. The overall response rate was 43% with 6 patients (26%) achieving complete remission (CR), 2 (9%) with CR with incomplete count recovery, and 2 (9%) with a morphologic leukemia-free state. Seven of 10 responders proceeded to allogeneic stem cell transplantation. The combination of selinexor with MEC is a feasibile treatment option for patients with R/R AML.

Post-Transplantation Cyclophosphamide-Based Graft-versus-Host Disease Prophylaxis.

BACKGROUND: In patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT), a calcineurin inhibitor plus methotrexate has been a standard prophylaxis against graft-versus-host disease (GVHD). A phase 2 study indicated the potential superiority of a post-transplantation regimen of cyclophosphamide, tacrolimus, and mycophenolate mofetil. METHODS: In a phase 3 trial, we randomly assigned adults with hematologic cancers in a 1:1 ratio to receive cyclophosphamide-tacrolimus-mycophenolate mofetil (experimental prophylaxis) or tacrolimus-methotrexate (standard prophylaxis). The patients underwent HSCT from an HLA-matched related donor or a matched or 7/8 mismatched (i.e., mismatched at only one of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci) unrelated donor, after reduced-intensity conditioning. The primary end point was GVHD-free, relapse-free survival at 1 year, assessed in a time-to-event analysis, with events defined as grade III or IV acute GVHD, chronic GVHD warranting systemic immunosuppression, disease relapse or progression, and death from any cause. RESULTS: In a multivariate Cox regression analysis, GVHD-free, relapse-free survival was significantly more common among the 214 patients in the experimental-prophylaxis group than among the 217 patients in the standard-prophylaxis group (hazard ratio for grade III or IV acute GVHD, chronic GVHD, disease relapse or progression, or death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P = 0.001). At 1 year, the adjusted GVHD-free, relapse-free survival was 52.7% (95% CI, 45.8 to 59.2) with experimental prophylaxis and 34.9% (95% CI, 28.6 to 41.3) with standard prophylaxis. Patients in the experimental-prophylaxis group appeared to have less severe acute or chronic GVHD and a higher incidence of immunosuppression-free survival at 1 year. Overall and disease-free survival, relapse, transplantation-related death, and engraftment did not differ substantially between the groups. CONCLUSIONS: Among patients undergoing allogeneic HLA-matched HSCT with reduced-intensity conditioning, GVHD-free, relapse-free survival at 1 year was significantly more common among those who received cyclophosphamide-tacrolimus-mycophenolate mofetil than among those who received tacrolimus-methotrexate. (Funded by the National Heart, Lung, and Blood Institute and others; BMT CTN 1703 ClinicalTrials.gov number, NCT03959241.).

Outcome prediction by the 2022 European LeukemiaNet genetic-risk classification for adults with acute myeloid leukemia: an Alliance study.

Recently, the European LeukemiaNet (ELN) revised its genetic-risk classification of acute myeloid leukemia (AML). We categorized 1637 adults with AML treated with cytarabine/anthracycline regimens according to the 2022 and 2017 ELN classifications. Compared with the 2017 ELN classification, 2022 favorable group decreased from 40% to 35% and adverse group increased from 37% to 41% of patients. The 2022 genetic-risk groups seemed to accurately reflect treatment outcomes in all patients and patients aged <60 years, but in patients aged ≥60 years, relapse rates, disease-free (DFS) and overall (OS) survival were not significantly different between intermediate and adverse groups. In younger African-American patients, DFS and OS did not differ between intermediate-risk and adverse-risk patients nor did DFS between favorable and intermediate groups. In Hispanic patients, DFS and OS did not differ between favorable and intermediate groups. Outcome prediction abilities of 2022 and 2017 ELN classifications were similar. Among favorable-risk patients, myelodysplasia-related mutations did not affect patients with CEBPAbZIP mutations or core-binding factor AML, but changed risk assignment of NPM1-mutated/FLT3-ITD-negative patients to intermediate. NPM1-mutated patients with adverse-risk cytogenetic abnormalities were closer prognostically to the intermediate than adverse group. Our analyses both confirm and challenge prognostic significance of some of the newly added markers.

Longitudinal Survival Outcomes in Allogeneic Stem Cell Transplantation: An Institutional Experience.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a potentially curative treatment for many hematological disorders, but is often complicated by relapse of the underlying disease, graft-versus-host disease (GVHD), and infectious complications. We conducted a retrospective analysis on patients undergoing allo-SCT from 1984 to 2018 to better understand how survival has changed longitudinally with therapeutic advancements made to mitigate these complications. Method: We analyzed data from 1943 consecutive patients who received allo-SCT. Patients were divided into groups (gps) based on the year (yr) of transplant. Primary endpoints were overall survival (OS), progression free survival (PFS), and GVHD-free relapse-free survival (GRFS). Secondary endpoints were the cumulative incidences of grade II−IV and grade III−IV acute GVHD (aGVHD), chronic GVHD (cGVHD), and non-relapse mortality (NRM). Results: Our study found statistically significant improvements in OS, PFS, and GRFS. Five-year PFS among the groups increased from 24% to 48% over the years. Five-year OS increased from 25% to 53%. Five-year GRFS significantly increased from 6% to 14%, but remained relatively unchanged from 2004 to 2018. Cumulative incidences of grade II−IV aGVHD increased since 2009 (p < 0.001). However, cumulative incidence of NRM decreased since 2004 (p < 0.001). Conclusions: Our data show improved OS, PFS, and GRFS post allo-SCT over decades. This may be attributed to advances in supportive care and treatments focused on mitigation of GVHD and relapse.

High early death rates, treatment resistance, and short survival of Black adolescents and young adults with AML.

Survival of patients with acute myeloid leukemia (AML) is inversely associated with age, but the impact of race on outcomes of adolescent and young adult (AYA; range, 18-39 years) patients is unknown. We compared survival of 89 non-Hispanic Black and 566 non-Hispanic White AYA patients with AML treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols. Samples of 327 patients (50 Black and 277 White) were analyzed via targeted sequencing. Integrated genomic profiling was performed on select longitudinal samples. Black patients had worse outcomes, especially those aged 18 to 29 years, who had a higher early death rate (16% vs 3%; P=.002), lower complete remission rate (66% vs 83%; P=.01), and decreased overall survival (OS; 5-year rates: 22% vs 51%; P<.001) compared with White patients. Survival disparities persisted across cytogenetic groups: Black patients aged 18 to 29 years with non-core-binding factor (CBF)-AML had worse OS than White patients (5-year rates: 12% vs 44%; P<.001), including patients with cytogenetically normal AML (13% vs 50%; P<.003). Genetic features differed, including lower frequencies of normal karyotypes and NPM1 and biallelic CEBPA mutations, and higher frequencies of CBF rearrangements and ASXL1, BCOR, and KRAS mutations in Black patients. Integrated genomic analysis identified both known and novel somatic variants, and relative clonal stability at relapse. Reduced response rates to induction chemotherapy and leukemic clone persistence suggest a need for different treatment intensities and/or modalities in Black AYA patients with AML. Higher early death rates suggest a delay in diagnosis and treatment, calling for systematic changes to patient care.

What is the role of leukopenia in the assessment of septic arthritis?

Background: It is not well-understood how leukopenia affects the synovial white blood cell (WBC) and percent neutrophils (%PMNs) in the setting of septic arthritis. We sought to determine 1. Do synovial WBC and %PMNs differ between patients with culture positive septic arthritis with or without leukopenia? And 2. Are traditional thresholds of synovial fluid studies for accurately diagnosing septic arthritis still applicable in the leukopenic patient population? Methods: A retrospective cohort study was performed at a single institution of 79 non-leukopenic and 11 leukopenic patients diagnosed with culture-positive septic arthritis. Demographic data, serum laboratory values, synovial laboratory values, and culture results were recorded. Significant differences in synovial laboratory values were evaluated using the Wilcoxon-Mann-Whitney test. Results are reported as median, interquartile range, and p values. Results: There was a significant difference in synovial WBC in leukopenic patients compared to non-leukopenic patients with culture positive septic arthritis (p = 0.01). No significant difference was found in the synovial %PMNs between two cohorts (p = 0.33). Conclusion: Leukopenic patients with culture positive septic arthritis have significantly lower synovial WBCs compared to non-leukopenic patients. Traditional thresholds for synovial WBC are not reliable for excluding diagnosis of septic arthritis in leukopenic patients.

Impact of Race and Geographic Area of Residence on Outcomes After Allogeneic Stem Cell Transplant.

Background: Allogeneic hematopoietic stem cell transplant (allo-HCT) is a potential curative therapy for a variety of hematologic disorders. However, it requires highly specialized care that is only available at select centers across the country. Thus, minority populations are at risk for healthcare disparities in access to and outcomes of allo-HCT. Our study aimed to assess the impact of race and location of residence on outcomes of allo-HCT. Methods: We performed a retrospective analysis of all patients who underwent allo-HCT at the Ohio State University from 1984 to 2018. Patients were divided by race (Caucasian, African American, and other) and grouped by zip code into rural, suburban, and urban groups. Primary endpoints included progression-free survival (PFS) and overall survival (OS). Results: Of the 1,943 patients included in the study, 94.3% self-identified as Caucasian, 4.6% African American, and 1.1% other. In total, 63.4% lived in rural areas, 22.9% suburban, and 13.8% urban. There was no significant difference in OS or PFS by race (p = 0.15, 0.21) or place of residence (p = 0.39, 0.17). In addition, no difference in nonrelapse mortality, acute and chronic graft-versus-host disease (GVHD), and GVHD-free relapse-free survival (GRFS) was seen among the race or place of residence. Conclusion: Our study suggests that when appropriate access to HCT is given, there is no difference in outcomes based on race, ethnicity or place of primary residence. Further research is needed to further evaluate barriers for these patients to undergo transplant and help mitigate these barriers.

Assessment for Septic Arthritis in Immunocompetent and Immunocompromised Patients: A Single-Institution Study.

INTRODUCTION: Prompt diagnosis of septic arthritis is imperative to prevent irreversible joint damage. Immunocompromised patients are at an increased risk of septic arthritis as well as secondary systemic infection. Our aims were to identify features predictive of septic arthritis and to determine whether these features differed between immunocompetent and immunocompromised patients. METHODS: A single institution retrospective cohort study was performed of 173 immunocompetent and 70 immunocompromised patients who underwent aspiration or arthrotomy for suspected septic arthritis from 2010 to 2018. Demographic data, symptoms, laboratory values, and imaging findings were recorded. Multiple variable logistic regression models were used to assess for predictive factors for septic arthritis in both cohorts. Results were reported as odds ratios, 95% confidence intervals, and P values. RESULTS: In the regression analysis, independent predictive factors for septic arthritis in immunocompetent patients were younger age (P = 0.004), presence of radiographic abnormalities (P = 0.006), and C-reactive protein (CRP) (P < 0.001). For immunocompromised patients, only CRP was an independent continuous predictive factor (P = 0.008) for septic arthritis. A risk stratification tool for predicting septic arthritis in immunocompetent patients using age <55 years, CRP >100 mg/dL, and presence of radiographic abnormalities was developed. A similar tool was created using CRP >180 mg/dL and radiographic abnormalities in immunocompromised patients. DISCUSSION: Differences in predictive factors for septic arthritis between immunocompromised and immunocompetent patients suggest dissimilar clinical presentations. The developed risk stratification tools allow one to predict the likelihood of septic arthritis in both groups. This may permit more accurate selection of patients for surgical intervention in the setting of insufficient data from synovial aspiration.

PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

Acute GVHD, BK virus hemorrhagic cystitis and age are risk factors for transplant-associated thrombotic microangiopathy in adults.

Hematopoietic cell transplantation-associated thrombotic microangiopathy (TMA) is a complication associated with higher nonrelapse mortality (NRM) in patients who undergo allogeneic transplant (HCT). Current classification criteria are not generally agreed on or validated, and the presence of confounding factors after transplant contribute to underdiagnosis or delayed diagnosis of TMA. We studied risk factors, incidence, and biomarkers of TMA in 119 adult allogeneic HCT recipients. Twenty-seven patients developed a clinically actionable phenotype of TMA (CA-TMA) and the incidence of CA-TMA was 22% by day 180. Among the 27 patients who developed CA-TMA, 10 developed it before the onset of acute graft-versus-host disease (aGVHD), and 17 patients developed it after the onset of aGVHD. We report for the first time that age >50 years, BK hemorrhagic cystitis, and other viral infections (CMV, HHV-6, or adenovirus) are risk factors for adult CA-TMA. Even after adjustment for aGVHD, CA-TMA was independently associated with significantly higher NRM. These data illustrate relationships between CA-TMA and aGVHD, describe new risk factors for CA-TMA and emphasizes the need to develop validated set of criteria for timely diagnosis.

Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant.

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/µg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

Outcomes of Bone Marrow Compared to Peripheral Blood for Haploidentical Transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) from a haploidentical (haplo) donor has emerged as a suitable alternative in the absence of a matched donor. However, haplo-HCT patients have a higher risk of graft-versus-host disease (GVHD). Hence, bone marrow (BM) stem cell source and post-transplant cyclophosphamide (PTCy) have been routinely used to help mitigate this. Due to ease of collection, peripheral blood (PB) stem cells are increasingly being considered for haplo-HCT. We retrospectively analyzed 74 patients (42 BM and 32 PB) who underwent haplo-HCT at Ohio State University from 2009 to 2018. Median age at transplant was 60 years (yrs) for BM and 54 yrs for PB, (p = 0.45). There was no difference in OS (p = 0.13) and NRM (p = 0.75) as well as PFS (p = 0.10) or GRFS (p = 0.90) between the groups. The BM cohort showed a 3-year OS rate of 63% (95% confidence interval (CI): 46-76), and 3-year PFS of 49% (95% CI: 33-63). For the PB group, 3-year OS and PFS were 78% (95% CI: 59-89) and 68% (95% CI: 49-82), respectively. There were no differences in the incidence of acute GVHD (grade II-IV) (p = 0.31) and chronic GVHD (p = 0.18). Patients receiving BM had a significantly higher risk for relapse with relapse rates by 2 years at 36% (95% CI: 22-50) vs. 16% (95% CI: 6-31) for PB (p = 0.03). The findings from this study suggest that PB is an excellent alternative to BM for haplo-HCT.

Inhibiting the Inhibitors of Apoptosis: When Two Targets Are Better Than One.

In this issue of Cancer Discovery, Pullarkat and colleagues present the results from a phase I clinical trial that is the first to combine small-molecule inhibitors for multiple antiapoptotic proteins, BCL2 as well as BCL-XL, with a traditional chemotherapy backbone for patients with relapsed/refractory acute lymphoblastic leukemia. This trial has demonstrated impressive response rates with acceptable toxicity while providing proof of concept that dual targeting-hitting BCL2 hard and BCL-XL soft-is both effective and tolerable in a heterogeneous patient population with prior existing cytopenias.See related article by Pullarkat et al., p. 1440.