Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Acute Myeloid Leukemia Causes T Cell Exhaustion and Depletion in a Humanized Graft-versus-Leukemia Model.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is, in many clinical settings, the only curative treatment for acute myeloid leukemia (AML). The clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. However, AML relapse remains the top cause of posttransplant death; this highlights the urgent need to enhance GVL. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. In this article, we report, the successful establishment of a novel (to our knowledge) humanized GVL model system by transplanting clinically paired donor PBMCs and patient AML into MHC class I/II knockout NSG mice. We observed significantly reduced leukemia growth in humanized mice compared with mice that received AML alone, demonstrating a functional GVL effect. Using this model system, we studied human GVL responses against human AML cells in vivo and discovered that AML induced T cell depletion, likely because of increased T cell apoptosis. In addition, AML caused T cell exhaustion manifested by upregulation of inhibitory receptors, increased expression of exhaustion-related transcription factors, and decreased T cell function. Importantly, combined blockade of human T cell-inhibitory pathways effectively reduced leukemia burden and reinvigorated CD8 T cell function in this model system. These data, generated in a highly clinically relevant humanized GVL model, not only demonstrate AML-induced inhibition of alloreactive T cells but also identify promising therapeutic strategies targeting T cell depletion and exhaustion for overcoming GVL failure and treating AML relapse after alloSCT.

The addition of doxycycline to fluoroquinolones for bacterial prophylaxis in autologous stem cell transplantation for multiple myeloma.

BACKGROUND: Bacterial prophylaxis with a fluoroquinolone (FQ) during autologous stem cell transplant (ASCT) is common, although not standardized among transplant centers. The addition of doxycycline (doxy) to FQ prophylaxis was previously linked to reduced neutropenic fever and bacteremia in multiple myeloma (MM) patients undergoing ASCT although several confounders were present. We compared the incidence of neutropenic fever and bacteremia between MM patients variably receiving prophylaxis with FQ alone and FQ-doxy during ASCT. METHODS: Systematic retrospective chart review of MM patients who underwent ASCT between January 2016 and December 2021. The primary objective was to determine the effect of bacterial prophylaxis on neutropenic fever and bacteremia within 30 days of ASCT. Multivariable logistic regression for neutropenic fever and univariate logistic regression for bacteremia accounted for differences in subject characteristics between groups. RESULTS: Among 341 subjects, 121 received FQ and 220 received FQ-doxy for prophylaxis. Neutropenic fever developed in 67 (55.4%) and 87 (39.5%) subjects in the FQ and FQ-doxy groups, respectively (p = .005). Bacteremia was infrequent, with 5 (4.1%) and 5 (2.3%) cases developing in the FQ and FQ-doxy groups, respectively (p = .337). Among Gram-negative bacteremia events, 7/7 Escherichia coli strains were FQ-resistant, and 5/7 were ceftriaxone-resistant. CONCLUSION: The FQ-doxy prophylaxis group had fewer cases of neutropenic fever than the FQ group, however, there was no significant difference in bacteremia. High rates of antibiotic resistance were observed. An updated randomized controlled trial investigating appropriate prophylaxis for ASCT in the context of current oncology standards and changing antimicrobial resistance rates is warranted.

Clofarabine Preconditioning followed by Allogeneic Transplant Using TBI and Post-Transplant Cyclophosphamide for Relapsed Refractory Leukemia.

Acute myeloid leukemia patients with induction failure or relapsed refractory disease have minimal chance of achieving remission with subsequent treatments. Several trials have shown the feasibility of clofarabine-based conditioning in allogeneic stem cell transplants (allo-HSCT) for non-remission AML patients. Pre-transplant conditioning with clofarabine followed by reduced-intensity allo-HSCT has also demonstrated a potential benefit in those patients with human leukocyte antigen (HLA)-identical donors, but it is not commonly used in haploidentical and mismatched transplants. In this case report, we describe our experience of seven cases of non-remission AML who received clofarabine preconditioning followed by an allo-HSCT with PTCy. The 2-year overall survival and disease-free survival was 83.3% (95% confidence interval (CI): 27.3-97.9%) and 85.7% (95% CI: 33.4-97.9%). Median days of neutrophil and platelet recovery were 16 (range of 13-23) and 28 (range of 17-75), respectively. The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) at day 100 and chronic GVHD at 1-year showed 28.6% (95% CI: 8-74.2%) and 28.6% (95% CI: 3-63.9%), respectively. The two-year relapse rate was 14.3% (95% CI: 2.14-66.6%). One-year GVHD-free relapse-free survival (GFRS) at 1-year was 71.4% (95% CI: 25.8-92%). Our patients showed successful outcomes with clofarabine preconditioning to reduce the leukemic burden at the pre-transplant period followed by PTCy to reduce GVHD resulting in lower relapsed rate and better GFRS in these patients.

Interleukin-4 treatment reduces leukemia burden in acute myeloid leukemia.

Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response, particularly in allergy and hypersensitivity. Interestingly, IL-4 increases endogenous levels of prostaglandin D2 (PGD2 ) and its metabolites, Δ12 -prostaglandin J2 (Δ12 -PGJ2 ) and 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ), collectively called cyclopentenone PGs (CyPGs). However, the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here, we employed a murine model of acute myeloid leukemia (AML), where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells, to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML, as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated, in part, by the enhanced expression of hematopoietic- PGD2  synthase (H-PGDS) to effect endogenous production of CyPGs, through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662, a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, suggested endogenous CyPGs-PPARγ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together, our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML.

Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Donor HLA mismatch promotes full donor T-cell chimerism in the allogeneic stem cell transplant with reduced-intensity conditioning and post-transplant cyclophosphamide GVHD prophylaxis.

Full donor T-cell chimerism (FDTCC) after allogeneic stem cell transplant (allo-SCT) has been associated with improved outcomes in hematologic malignancy. We studied if donor human leukocyte antigen (HLA) mismatch improves achievement of FDTCC because mismatched HLA promotes donor T-cell proliferation where recipient T-cells had been impaired by previous treatment. Patients (N = 138) received allo-SCT with reduced-intensity conditioning (RIC) from 39 HLA mismatched donors (16 unrelated; 23 haploidentical) with post-transplant cyclophosphamide (PTCy) or 99 matched donors (21 siblings; 78 unrelated) with PTCy (N = 18) or non-PTCy (N = 81). Achievement of FDTCC by day 100 was higher with HLA mismatched donors than matched donors (82.1% vs. 27.3%, p < 00,001), which was further improved with 200 cGy total body irradiation (87.9%) or lymphoid (versus myeloid) malignancy (93.8%). Since all mismatched transplants used PTCy, FDTCC was higher with PTCy than non-PTCy (68.4% vs. 25.7%, p < 0.00001), but not in the matched transplant with PTCy (38.9%), negating PTCy as the primary driver. Lymphocyte recovery was delayed with PTCy than without (median on day + 30: 100 vs. 630/µL, p < 0.0001). The benefit of FDTCC was not translated into survival outcomes, especially in myeloid malignancies, possibly due to the insufficient graft-versus-tumor effects from the delayed lymphocyte recovery. Further studies are necessary to improve lymphocyte count recovery in PTCy transplants.

Glucocorticoids enhance the antileukemic activity of FLT3 inhibitors in FLT3-mutant acute myeloid leukemia.

FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.

Therapeutic Benefits of Selenium in Hematological Malignancies.

Supplementing chemotherapy and radiotherapy with selenium has been shown to have benefits against various cancers. This approach has also been shown to alleviate the side effects associated with standard cancer therapies and improve the quality of life in patients. In addition, selenium levels in patients have been correlated with various cancers and have served as a diagnostic marker to track the efficiency of treatments or to determine whether these selenium levels cause or are a result of the disease. This concise review presents a survey of the selenium-based literature, with a focus on hematological malignancies, to demonstrate the significant impact of selenium in different cancers. The anti-cancer mechanisms and signaling pathways regulated by selenium, which impart its efficacious properties, are discussed. An outlook into the relationship between selenium and cancer is highlighted to guide future cancer therapy development.

Results of a randomized phase 3 study of oral sapacitabine in elderly patients with newly diagnosed acute myeloid leukemia (SEAMLESS).

BACKGROUND: Acute myeloid leukemia (AML) is fatal in elderly patients who are unfit for standard induction chemotherapy. The objective of this study was to evaluate the survival benefit of administering sapacitabine, an oral nucleoside analogue, in alternating cycles with decitabine, a low-intensity therapy, to elderly patients with newly diagnosed AML. METHODS: This randomized, open-label, phase 3 study (SEAMLESS) was conducted at 87 sites in 11 countries. Patients aged ≥70 years who were not candidates for or chose not to receive standard induction chemotherapy were randomized 1:1 to arm A (decitabine in alternating cycles with sapacitabine) received 1-hour intravenous infusions of decitabine 20 mg/m2 once daily for 5 consecutive days every 8 weeks (first cycle and subsequent odd cycles) and sapacitabine 300 mg twice daily on 3 consecutive days per week for 2 weeks every 8 weeks (second cycle and subsequent even cycles) or to control arm C who received 1-hour infusions of decitabine 20 mg/m2 once daily for 5 consecutive days every 4 weeks. Prior hypomethylating agent therapy for preexisting myelodysplastic syndromes or myeloproliferative neoplasms was an exclusion criterion. Randomization was stratified by antecedent myelodysplastic syndromes or myeloproliferative neoplasms, white blood cell count (<10 × 109 /L and ≥10 × 109 /L), and bone marrow blast percentage (≥50% vs <50%). The primary end point was overall survival (OS). Secondary end points were the rates of complete remission (CR), CR with incomplete platelet count recovery, partial remission, hematologic improvement, and stable disease along with the corresponding durations, transfusion requirements, number of hospitalized days, and 1-year survival. The trial is registered at ClinicalTrials.gov (NCT01303796). RESULTS: Between October 2011 and December 2014, 482 patients were enrolled and randomized to receive decitabine administered in alternating cycles with sapacitabine (study arm, n = 241) or decitabine monotherapy (control arm, n = 241). The median OS was 5.9 months on the study arm versus 5.7 months on the control arm (P = .8902). The CR rate was 16.6% on the study arm and 10.8% on the control arm (P = .1468). In patients with white blood cell counts <10 × 109 /L (n = 321), the median OS was higher on the study arm versus the control arm (8.0 vs 5.8 months; P = .145), as was the CR rate (21.5% vs 8.6%; P = .0017). CONCLUSIONS: The regimen of decitabine administered in alternating cycles with sapacitabine was active but did not significantly improve OS compared with decitabine monotherapy. Subgroup analyses suggest that patients with baseline white blood cell counts <10 × 109 /L might benefit from decitabine alternating with sapacitabine, with an improved CR rate and the convenience of an oral drug. These findings should be prospectively confirmed.

A pediatric regimen for older adolescents and young adults with acute lymphoblastic leukemia: results of CALGB 10403.

Retrospective studies have suggested that older adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL) have better survival rates when treated using a pediatric ALL regimen administered by pediatric treatment teams. To address the feasibility and efficacy of using a pediatric treatment regimen for AYA patients with newly diagnosed ALL administered by adult treatment teams, we performed a prospective study, CALGB 10403, with doses and schedule identical to those in the Children's Oncology Group study AALL0232. From 2007 to 2012, 318 patients were enrolled; 295 were eligible and evaluable for response. Median age was 24 years (range, 17-39 years). Use of the pediatric regimen was safe; overall treatment-related mortality was 3%, and there were only 2 postremission deaths. Median event-free survival (EFS) was 78.1 months (95% confidence interval [CI], 41.8 to not reached), more than double the historical control of 30 months (95% CI, 22-38 months); 3-year EFS was 59% (95% CI, 54%-65%). Median overall survival (OS) was not reached. Estimated 3-year OS was 73% (95% CI, 68%-78%). Pretreatment risk factors associated with worse treatment outcomes included obesity and presence of the Philadelphia-like gene expression signature. Use of a pediatric regimen for AYAs with ALL up to age 40 years was feasible and effective, resulting in improved survival rates compared with historical controls. CALGB 10403 can be considered a new treatment standard upon which to build for improving survival for AYAs with ALL. This trial was registered at www.clinicaltrials.gov as #NCT00558519.

Improved outcome in AML relapse after allogeneic transplant with high-intensity chemotherapy followed by 2nd allogeneic stem cell transplant or donor lymphocyte infusion.

Acute myeloid leukemia (AML) relapse after allogeneic stem cell transplant (alloSCT) remains a major therapeutic challenge. While patients with longer remission after initial alloSCT are recommended to receive cell therapy (CT) such as 2ndalloSCT or donor lymphocyte infusion (DLI), survival for patients who relapse within 6 months of alloSCT has been dismal. We evaluated the outcomes of AML relapse after alloSCT to assess the impact of different treatments on long-term survival. One hundred and seventy-two patients with AML underwent alloSCT at the Penn State Cancer Institute from January 2014 to August 2019. Sixty-nine patients relapsed (median age, 60 years; range, 10-75). Of these, 4 patients underwent 2ndalloSCT, and 26 received DLI. One-year overall survival (OS) in all cases was 20.3% (95% CI: 11.8-30.4%). Patients with ECOG performance status (PS) 0-2 at relapse showed a better 1-year OS than those with PS 3-4. Median OS for patients who received chemotherapy only or chemotherapy with CT was 74 or 173.5 days, respectively (p < 0.001). Relapsed patients receiving conventional re-induction chemotherapy were categorized as the high-intensity chemotherapy (H) group, while those receiving treatments such as hypomethylating agents or targeted agents were categorized as the low-intensity chemotherapy (L) group. The H group showed a better 1-year OS compared with the L group. Patients who received H + CT showed a better 1-year OS of 52.9% than the other 3 groups (p < 0.001). Even for patients with post-alloSCT remission duration of less than 6 months, the statistical significance was preserved. Factors including age, donor source at 1stalloSCT, time to relapse, blast counts, PS at relapse, and treatment type after post-alloSCT relapse were used for a multivariate analysis, and matched or mismatched related donor and H + CT after alloSCT were identified as independent factors associated with OS. These findings support the use of H + CT as the treatment option of choice for AML patients who relapse after alloSCT when feasible.

Multi-dimensional analysis identifies an immune signature predicting response to decitabine treatment in elderly patients with AML.

Decitabine is a DNA-hypomethylating agent that has been widely applied for the treatment of acute myeloid leukaemia (AML) patients who are elderly or unfit for intensive therapy. Although effective, the complete response rate to decitabine is only around 30% and the overall survival remains poor. Emerging data support that regulation of DNA methylation is critical to control immune cell development, differentiation and activation. We hypothesize that defining how decitabine influences the immune responses in AML will facilitate the development of novel immune-based leukaemia therapeutics. Here, we performed phenotypic and functional immune analysis on clinical samples from AML patients receiving decitabine treatment and demonstrated a significant impact of decitabine on the immune system. T-cell expression of inhibitory molecules was upregulated and the ability of CD8 T cells to produce cytokines was decreased upon decitabine treatment. Importantly, in an unbiased comprehensive analysis, we identified a unique immune signature containing a cluster of key immune markers that clearly separate patients who achieved complete remission after decitabine from those who failed to do so. Therefore, this immune signature has a strong predictive value for clinical response. Collectively, our study suggests that immune-based analyses may predict clinical response to decitabine and provide a therapeutic strategy to improve the treatment of AML.

The novel Isatin analog KS99 targets stemness markers in acute myeloid leukemia.

Leukemic stem cells are multipotent, self-renewing, highly proliferative cells that can withstand drug treatments. Although currently available treatments potentially destroy blast cells, they fail to eradicate leukemic progenitor cells completely. Aldehyde dehydrogenase and STAT3 are frequently up-regulated in pre-leukemic stem cells as well as in acute myeloid leukemia (AML) expressing the CD34+CD38- phenotype. The Isatin analog, KS99 has shown anticancer activity against multiple myeloma which may, in part, be mediated by inhibition of Bruton's tyrosine kinase activation. Here we demonstrate that KS99 selectively targets leukemic stem cells with high aldehyde dehydrogenase activity and inhibits phosphorylation of STAT3. KS99 targeted cells co-expressing CD34, CD38, CD123, TIM-3, or CD96 immunophenotypes in AML, alone or in combination with the standard therapeutic agent cytarabine. AML with myelodysplastic-related changes was more sensitive than de novo AML with or without NPM1 mutation. KS99 treatment reduced the clonogenicity of primary human AML cells as compared to normal cord blood mononuclear cells. Downregulation of phosphorylated Bruton's tyrosine kinase, STAT3, and aldehyde dehydrogenase was observed, suggesting interaction with KS99 as predicted through docking. KS99 with or without cytarabine showed in vivo preclinical efficacy in human and mouse AML animal models and prolonged survival. KS99 was well tolerated with overall negligible adverse effects. In conclusion, KS99 inhibits aldehyde dehydrogenase and STAT3 activities and causes cell death of leukemic stem cells, but not normal hematopoietic stem and progenitor cells.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Acid ceramidase promotes drug resistance in acute myeloid leukemia through NF-κB-dependent P-glycoprotein upregulation.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

Phase 1-2 study of vorinostat (SAHA), cladribine and rituximab (SCR) in relapsed B-cell non-Hodgkin lymphoma and previously untreated mantle cell lymphoma.

Altered DNA methylation and histone acetylation in lymphoma provided the rationale for using vorinostat (SAHA), cladribine and rituximab (SCR) in non-Hodgkin lymphomas (NHL) in this phase 1-2 study (NCT00764517). Treatment included cladribine 5 mg/m2 intravenously (IV) (days 1-5), rituximab 375 mg/m2 IV (weekly 4× for cycle 1 and 1×/month) and vorinostat orally once daily (days 1-14) every 28 days for up to six cycles. Phase 1 included relapsed patients (n = 10) in a standard 3 + 3 dose escalation design (vorinostat: 200, 300 and 400 mg). No dose-limiting toxicities were seen. The phase 2 dose for vorinostat was 400 mg po (days 1-14). The majority of phase 2 patients had mantle cell lymphoma (MCL) (n = 57; 39 previously untreated, 10 relapsed). The primary objective was objective response rate [complete response (CR) + partial response] which was 39% (7/18) in relapsed patients and 97% (38/39) with 80% (31/39) attaining a CR in previously untreated MCL. At a median follow-up of 42 months, median progression-free survival (PFS) and overall survival (OS) for relapsed NHL were 19·5 [95% confidence interval (CI): 2·0-33·0] and 25·0 (95% CI: 12·0-45·0) months respectively. Median PFS for previously untreated MCL was 84·0 months; OS could not be estimated. Toxicities were primarily haematological.

Pentostatin, Cyclophosphamide, and Rituximab Followed by Alemtuzumab for Relapsed or Refractory Chronic Lymphocytic Leukemia: A Phase 2 Trial of the ECOG-Acrin Cancer Research Group (E2903).

Patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) may benefit from salvage chemoimmunotherapy (CIT). To explore further the use of CIT in the pre-novel agent era, ECOG-ACRIN undertook a phase 2 trial (E2903) for R/R CLL utilizing pentostatin, cyclophosphamide, and rituximab (PCR) followed by a consolidation course of alemtuzumab. This trial enrolled 102 patients with a median age of 64 years. Treatment consisted of 6 cycles of PCR followed by alemtuzumab for either 4 or 18 weeks depending on the initial response to PCR. The overall response after PCR (complete remission, CR, nodular partial remission, nPR, and partial remission, PR) was 55%. Major responses (CR or nPR) were achieved in 6%. The median overall survival (OS) and the median progression-free survival were 28 and 12 months, respectively. The most serious nonlethal adverse events were myelosuppression, febrile neutropenia, fatigue, nausea, and hyponatremia. PCR is an effective and well-tolerated nucleoside-based regimen for heavily pretreated CLL patients with R/R disease. The addition of alemtuzumab to CLL patients with a minor response (PR) or stable disease did not result in a significant number of higher responses (CR or nPR) nor an improvement in OS.

Outcomes of CLL patients treated with sequential kinase inhibitor therapy: a real world experience.

B-cell receptor kinase inhibitor (KI) therapy represents a paradigm shift in chronic lymphocytic leukemia (CLL) management, but data on practice patterns after KI discontinuation and optimal sequencing are limited. We conducted a multicenter, retrospective, comprehensive analysis on 178 patients with CLL (ibrutinib = 143; idelalisib = 35) who discontinued KI therapy. We examined responses, toxicity, post-KI therapies, and overall survival (OS). Patients had a median of 3 prior therapies (range 0-11); del17p (34%), p53 mutation (27%), del11q (33%), and complex karyotype (29%). Overall response rate (ORR) to first KI was 62% (complete response 14%). The most common reasons for KI discontinuation were toxicity (51%), CLL progression (29%), and Richter transformation (RT) (8%). Median progression-free survival (PFS) and OS from KI initiation were 10.5 and 29 months, respectively. Notably, initial KI choice did not impact PFS or OS; however, RT portended significantly inferior OS (P = .0007). One hundred fourteen patients received subsequent salvage therapy following KI discontinuation with an ORR to subsequent KI at 50% and a median PFS of 11.9 months. Median PFS in KI-intolerant patients treated with an alternate KI was not reached vs 7 months for patients with CLL progression. In summary, these data demonstrate that toxicity was the most common reason for KI discontinuation, that patients who discontinue KI due to toxicity can respond to an alternate KI, and that these responses may be durable. This trial was registered at www.clinicaltrials.gov as #NCT02717611 and #NCT02742090.