Prognostic significance of myeloid-derived suppressor cells and systemic inflammation in newly diagnosed diffuse large B cell lymphoma treated with chemoimmunotherapy.

The revised International Prognostic Index (R-IPI) is an important prognostic tool in diffuse large B cell lymphoma (DLBCL); however, outcomes can vary markedly within R-IPI groups, and additional prognostic markers are needed. We conducted a prospective observational study to evaluate the circulating immature myeloid (IM) cell subsets and cytokine profiles of 31 patients with newly diagnosed DLBCL before and after chemoimmunotherapy. Among circulating IM cells, myeloid-derived suppressor cells (MDSCs) were the predominant cell type (73.8% ± 26%). At baseline, circulating monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) were predominantly mutually exclusive. Patients with DLBCL clustered into three distinct immunotypes according to MDSC levels and subtype predominance: M-MDSChigh, PMN-MDSChigh, and MDSClow. The M-MDSChigh immunotype was associated with the germinal center B cell-like (GCB) subtype and elevated serum IL-8 and MIP-1α levels. PMN-MDSChigh was associated with the non-GCB subtype and elevated IL-8, MCP-1, IP-10, TNFα, and IL-1Ra levels. Standard chemoimmunotherapy partially reduced M-MDSC distribution across the MDSClow and M-MDSChigh groups. By contrast, among the MDSClow and PMN-MDSChigh groups, PMN-MDSCs persisted after treatment. Two high-risk patients with non-GCB DLBCL and MDSClow immunotype experienced early disease recurrence within 12 months of treatment completion. This study demonstrates that distinct types of MDSCs are associated with subtypes of DLBCL. MDSC levels are dynamic and may be associated with disease status. Persistence of PMN-MDSCs among high-risk patients with DLBCL may be associated with early relapse.

Pharmacogenetic and clinical predictors of voriconazole concentration in hematopoietic stem cell transplant recipients receiving CYP2C19-guided dosing.

CYP2C19-guided voriconazole dosing reduces pharmacokinetic variability, but many patients remain subtherapeutic. The aim of this study was to evaluate the effect of candidate genes and a novel CYP2C haplotype on voriconazole trough concentrations in patients receiving CYP2C19-guided dosing. This is a retrospective candidate gene study in allogeneic hematopoietic cell transplant (HCT) patients receiving CYP2C19-guided voriconazole dosing. Patients were genotyped for ABCB1, ABCG2, CYP2C9, CYP3A4, CYP3A5, and the CYP2C haplotype. Of 185 patients, 36% were subtherapeutic (of which 79% were normal or intermediate metabolizers). In all patients, CYP2C19 (p < 0.001), age (p = 0.018), and letermovir use (p = 0.001) were associated with voriconazole concentrations. In the subset receiving 200 mg daily (non-RM/UMs), CYP2C19 (p = 0.004) and ABCG2 (p = 0.015) were associated with voriconazole concentrations; CYP2C19 (p = 0.028) and letermovir use (p = 0.001) were associated with subtherapeutic status. CYP2C19 phenotype and letermovir use were significantly associated with subtherapeutic voriconazole concentrations and may be used to improve voriconazole precision dosing, while further research is needed to clarify the role of ABCG2 in voriconazole dosing.

A Clinical and Correlative Study of Elotuzumab, Carfilzomib, Lenalidomide, and Dexamethasone (Elo-KRd) for Lenalidomide Refractory Multiple Myeloma in First Relapse.

INTRODUCTION: Treatment of patients with multiple myeloma (MM) in first relapse remains a challenge. This phase II study combined elotuzumab (Elo) with carfilzomib, lenalidomide, and dexamethasone (KRd) for treatment of MM in first relapse with the aim of improving efficacy. METHODS: Enrolled patients received Elo-KRd induction for 4 cycles, and Elo-lenalidomide maintenance until progression. The primary endpoint was VGPR or better (≥VGPR) postinduction. Secondary endpoints were MRD by flow cytometry, OS, PFS, and safety. Correlatives included characterization of the impact of Elo-KRd on NK and T cell subsets via flow cytometry. Target accrual of 40 patients was not met due to COVID-19 pandemic. RESULTS: Of 15 patients enrolled, 10 (67%) had high-risk features (del17p, t[4;14], t[14;16], 1q gain/amplification, plasma cell leukemia, extramedullary MM, or functional high risk), 12 (80%) were lenalidomide-refractory, and 5 (33.3%) bortezomib-refractory. Postinduction ≥VGPR was 7/15 (46.7%) and MRD-negative (10-5) rate 20%. Overall response during study was 80%, including ≥VGPR as best response of 53.3%. At median follow-up of 28.2 (range, 3.8 to 44.2) months, the median PFS was 11.5 months (95% CI 1.9, 18), and median OS not reached (95% CI 10.1, NA). No new safety concerns were reported. Elo-KRd treatment did not augment NK cell distribution or activity in blood or bone marrow. Effector CD4+ and CD8+ T cells significantly decreased postinduction, with concomitant acquisition of T central memory phenotype, particularly at a high rate in ≥VGPR group. CONCLUSION: A short course of Elo-KRd induction followed by Elo-lenalidomide maintenance demonstrated activity in predominantly lenalidomide-refractory and / or high-risk MM. The results with this well-tolerated combination are comparable to other contemporary approved triplet combinations.

Cost saving, patient centered algorithm for progenitor cell mobilization for autologous hematopoietic cell transplantation.

Administration of plerixafor with granulocyte-colony stimulating factor (G-CSF) mobilizes CD34+ cells much more effectively than G-CSF alone, but cost generally limits plerixafor use to patients at high risk of insufficient CD34+ cell collection based on low peripheral blood (PB) CD34+ counts following 4 days of G-CSF. We analyzed costs associated with administering plerixafor to patients with higher day 4 CD34+ cell counts to decrease apheresis days and explored the use of a fixed split dose of plerixafor instead of weight-based dosing. We analyzed 235 patients with plasma cell disorders or non-Hodgkin's lymphoma who underwent progenitor cell mobilization and autologous hematopoietic cell transplantation (AHCT) between March 2014 and December 2017. Two hundred ten (89%) received G-CSF plus Plerixafor and 25 (11%) received G-CSF alone. Overall, 180 patients (77%) collected in 1 day, 53 (22%) in 2 days and 2 (1%) in 3 days. Based on our data, we present a probabilistic algorithm to identify patients likely to require more than one day of collection using G-CSF alone. CD34+ cell yield, ANC and platelet recovery were not significantly different between fixed and standard dose plerixafor. Plerixafor enabled collection in 1 day and with estimated savings of $5000, compared to patients who did not receive plerixafor and required collection for three days. While collection and processing costs and patient populations vary among institutions, our results suggest re-evaluation of current algorithms.

Heterozygous germ line CSF3R variants as risk alleles for development of hematologic malignancies.

Colony-stimulating factor 3 receptor (CSF3R) encodes the receptor for granulocyte colony-stimulating factor (G-CSF), a cytokine vital for granulocyte proliferation and differentiation. Acquired activating heterozygous variants in CSF3R are the main cause of chronic neutrophilic leukemia, a hyperproliferative disorder. In contrast, biallelic germ line hypomorphic variants in CSF3R are a rare cause of severe congenital neutropenia, a hypoproliferative condition. The impact of heterozygous germ line CSF3R variants, however, is unknown. We identified CSF3R as a new germ line hematologic malignancy predisposition gene through analysis of 832 next-generation sequencing tests conducted in 632 patients with hematologic malignancies. Among germ line CSF3R variants, 3 were abnormal in functional testing, indicating their deleterious nature. p.Trp547* was identified in 2 unrelated men with myelodysplastic syndromes diagnosed at 76 and 33 years of age, respectively. p.Trp547* is a loss-of-function nonsense variant in the extracellular domain that results in decreased CSF3R messenger RNA expression and abrogation of CSF3R surface expression and proliferative responses to G-CSF. p.Ala119Thr is a missense variant found in 2 patients with multiple myeloma and acute lymphoblastic leukemia, respectively. This variant is located between the extracellular immunoglobulin-like and cytokine receptor homology domains and results in decreased G-CSF sensitivity. p.Pro784Thr was identified in a 67-year-old man with multiple myeloma. p.Pro784Thr is a missense variant in the cytoplasmic domain that inhibits CSF3R internalization, producing a gain-of-function phenotype and G-CSF hypersensitivity. Our findings identify germ line heterozygous CSF3R variants as risk factors for development of myeloid and lymphoid malignancies.

Altered expression of CSF3R splice variants impacts signal response and is associated with SRSF2 mutations.

Three annotated CSF3R mRNA splice variants have been described. CSF3R-V1 is the wild-type receptor, while CSF3R-V4 is a truncated form increased in some patients with AML. CSF3R-V3 mRNA was identified in placenta more than 20 years ago, but remains largely uncharacterized due to the lack of a suitable detection assay. Using a novel digital PCR method to quantitate expression of each CSF3R mRNA splice variant in hematopoietic cells, CSF3R-V1 was most highly expressed followed by CSF3R-V3. Functional assays revealed expression of V3 alone conferred a hypoproliferative phenotype associated with defective JAK-STAT activation. However, coexpression of V1 with V3 rescued proliferative responses. Comparative analysis of V3/V1 expression in CD34+ cells from healthy donors and patients with AML revealed a statistically significant increase in the V3/V1 ratio only in the subset of patients with AML harboring SRSF2 mutations. Knockout of SRFS2 in KG-1 and normal CD34+ cells decreased the V3/V1 ratio. Collectively, these data are the first to demonstrate expression of the CSF3R-V3 splice variant in primary human myeloid cells and a role for SRSF2 in modulating CSF3R splicing. Our findings provide confirmatory evidence that CSF3R is a target of SRSF2 mutations, which has implications for novel treatment strategies for SRSF2-mutated myeloid malignancies.

T618I CSF3R mutations in chronic neutrophilic leukemia induce oncogenic signals through aberrant trafficking and constitutive phosphorylation of the O-glycosylated receptor form.

Activating mutations in the membrane-proximal region of the colony-stimulating factor 3 receptor (CSF3R) are a hallmark of chronic neutrophilic leukemia (CNL) with the T618I mutation being most common. The mechanisms underlying constitutive activation of the T618I CSF3R and its signal propagation are poorly understood. Ligand-independent activation of the T618I CSF3R has previously been attributed to loss of receptor O-glycosylation and increased receptor dimerization. Here, we show that the T618I CSF3R is indeed glycosylated but undergoes enhanced spontaneous internalization and degradation that results in a marked decrease in its surface expression. Inhibition of the proteasome dramatically increases expression of the O-glycosylated T618I CSF3R. We also demonstrate that the O-glycosylated wild-type CSF3R is tyrosine phosphorylated in response to ligand but constitutively phosphorylated in cells expressing T618I CSF3R. Constitutive tyrosine phosphorylation of the O-glycosylated T618I receptor form correlated with activation of JAK2 and both the mutant receptor and JAK2 were found to be constitutively ubiquitinated. These observations provide novel insights into the mechanisms of oncogenic signaling by T618I CSF3R mutations in CNL.

CXCL12-CXCL4 heterodimerization prevents CXCL12-driven breast cancer cell migration.

Despite improvements in cancer early detection and treatment, metastatic breast cancer remains deadly. Current therapeutic approaches have very limited efficacy in patients with triple negative breast cancer. Among the many mechanisms associated that contribute to cancer progression, signaling through the CXCL12-CXCR4 is an essential step in cancer cell migration. We previously demonstrated the formation of CXCL12-CXCL4 heterodimers (Carlson et al., 2013). Here, we investigated whether CXCL12-CXCL4 heterodimers alter tumor cell migration. CXCL12 alone dose-dependently promoted the MDA-MB 231 cell migration (p < .05), which could be prevented by blocking the CXCR4 receptor. The addition of CXCL4 inhibited the CXCL12-induced cell migration (p < .05). Using NMR spectroscopy, we identified the CXCL4-CXCL12 binding interface. Moreover, we generated a CXCL4-derived peptide homolog of the binding interface that mimicked the activity of native CXCL4 protein. These results confirm the formation of CXCL12-CXCL4 heterodimers and their inhibitory effects on the migration of breast tumors cells. These findings suggest that specific peptides mimicking heterodimerization of CXCL12 might prevent breast cancer cell migration.

Effect of CYP3A4, CYP3A5, and ABCB1 Polymorphisms on Intravenous Tacrolimus Exposure and Adverse Events in Adult Allogeneic Stem Cell Transplant Patients.

Pharmacogenetics influences oral tacrolimus exposure; however, little data exist regarding i.v. tacrolimus. We investigated the impact of genetic polymorphisms in CYP3A4, CYP3A5, and ABCB1 on i.v. tacrolimus exposure and toxicity in adult patients receiving an allogeneic hematopoietic stem cell transplant for hematologic malignancies. Germline DNA was extracted from buccal swabs and genotyped for CYP3A4, CYP3A5, and ABCB1 polymorphisms. Continuous i.v. infusion of tacrolimus .03 mg/kg/day was initiated on day +5 post-transplant, and steady-state blood concentrations were measured 4days later. We evaluated the association between phenotypes and prevalence of nontherapeutic target concentrations (below or above 5 to 15 ng/mL) as well as tacrolimus-related toxicities. Of 63 patients, 28.6% achieved the target concentration; 71.4% were >15ng/mL, which was more common in CYP3A4 intermediate/normal metabolizers (compared with rapid) and those with at least 1 ABCB1 C2677T loss-of-function allele (P < .05). ABCB1 C2677T was significantly associated with concentrations >15ng/mL (odds ratio, 6.2; 95% confidence interval, 1.8 to 23.6; P = .004) and tacrolimus-related toxicities (odds ratio, 7.5; 95% confidence interval, 1.6 to 55.2; P = .02). ABCB1 C2677T and CYP3A4 are important determinants of i.v. tacrolimus exposure, whereas ABCB1 C2677T also impacts tacrolimus-related toxicities in stem cell transplants.

Peripheral Immunotype Correlates with Minimal Residual Disease Status and Is Modulated by Immunomodulatory Drugs in Multiple Myeloma.

Data indicate reversal of immune dysfunction with active treatment; however, the precise contribution of specific immune effector and immune suppressor components to achieve a minimal residual disease (MRD) state and immunomodulatory drug-mediated immunomodulatory effects in multiple myeloma (MM) patients remains poorly understood. In this prospective proof-of-principle study we sought to determine the dynamic alterations in natural killer (NK), NK-T, and T cells, including maturation and activating/inhibitory repertoire associated with MRDpos versus MRDneg status after autologous stem cell transplantation (ASCT) and during lenalidomide-based maintenance therapy. Of the 46MM patients enrolled, 36 had bone marrow MRD assessment 60+ days post-ASCT, 30 had longitudinal blood immunotyping during maintenance (pretherapy and after cycles 1, 3, and 6), and 20 had both MRD assessment and longitudinal immunotyping. Multicolor flow cytometry was used for MRD and immunotyping. Although the absolute number of NK cells was significantly lower in patients with MRDpos response, phenotypically NK cells in these patients displayed higher expression of activating receptors KIRDS4 and decreased expression of inhibitory molecules NKG2A compared with the MRDneg group. Furthermore, we observed significantly lower frequencies of T cells displaying KIR3DL1 in MRDpos versus MRDneg patients. Longitudinal immunotyping during lenalidomide maintenance showed loss of mature NK effector function, augmentation of NK-T effector function, and acquisition of PD1 independent anergic state. Our findings also suggest skewing of T cells toward an exhausted state during the maintenance phase in MRDpos patients. Put together, these observations provide a distinctive signature for MRDneg and MRDpos groups. These data support exploration of immune profiling in prospective clinical trials according to MRD-defined responses to identify patients that may benefit from maintenance intensification/modification or maintenance withdrawal.

Deep sustained response to daratumumab monotherapy associated with T-cell expansion in triple refractory myeloma.

BACKGROUND: Daratumumab, a human CD38 monoclonal antibody that has direct on-tumor and immunomodulatory mechanisms of action, demonstrated clinical benefit as monotherapy or in combination with established regimens in patients with multiple myeloma with one or more prior lines of therapy. CASE PRESENTATION: A male patient, who was 70 years of age at the time of diagnosis of multiple myeloma in 2011, relapsed after five lines of therapy, including autologous stem cell transplantation. The patient's disease, which was considered high risk with a deletion of chromosome 17p, advanced quickly and was triple refractory 2 years after diagnosis leaving few treatment options. He was treated with daratumumab monotherapy in the SIRIUS clinical trial resulting in a stringent complete response and clearance of minimal residual disease. The duration of the patient's clinical response is now over 3.5 years without relapse, compared with a median of 7.6 months for similarly treated patients. The patient's immunophenotype revealed CD8+ T-cell expansion, clonal expansion of the T-cell receptor repertoire, and decreases in regulatory T cells during daratumumab therapy, suggesting a robust adaptive immune response. This immune response was still present 32 months into daratumumab therapy. CONCLUSIONS: The results from this case report showed that a patient with advanced multiple myeloma, who had exhausted all treatment options with existing regimens, mounted an ongoing, deep, and durable response to daratumumab monotherapy. Further investigation of the immunologic profile provided additional patient-level evidence of an immunomodulatory mechanism of action of daratumumab.Trial registration ClinicalTrials.gov Identifier number NCT01985126. Submitted 22 July 2013.

Investigation of a gene signature to predict response to immunomodulatory derivatives for patients with multiple myeloma: an exploratory, retrospective study using microarray datasets from prospective clinical trials.

BACKGROUND: Immunomodulatory derivatives (IMiDs), along with proteasome inhibitors, are key components of treatment regimens for multiple myeloma. Nonetheless, outcomes vary among treated individuals. Drug-specific gene-expression profile (GEP) signatures that aid the prediction of favourable and unfavourable outcomes can provide patients with the most effective therapy for their individual disease. We aimed to develop and validate a gene expression signature to suggest which patients would benefit most from IMiD-based therapies. METHODS: For this exploratory retrospective study, we selected a cohort of patients with newly diagnosed or relapsed or refractory multiple myeloma who were treated in clinical trials with IMiD-containing regimens. Cohorts were eligible if they had publicly available GEP data from patients' bone marrow plasma cells, with long-term follow-up and clinicopathological data. In the development stage of the model, we identified 176 IMiD response genes that were differentially expressed before and after IMiD exposure using pharmacogenomic GEP data from patients who had bone marrow samples taken before and 48 h after a test dose exposure with thalidomide (n=42), lenalidomide (n=18), or pomalidomide (n=18). 14 of these genes had p values less than 0·05 for associations with progression-free survival in patients who received thalidomide in induction and maintenance therapy in the Total Therapy (TT) 2 trial (ie, the training cohort). We combined the 14 genes to create a continuous IMiD-14 score and an optimal cutoff. The subgroup with an IMiD-14 score higher than the cutoff was deemed to be IMiD-resistant. We obtained validation cohorts from four studies of IMiD combination regimens: the TT3a trial (thalidomide in induction and maintenance), the TT3b trial (thalidomide in induction and lenalidomide in maintenance), the TT6 trial (thalidomide in induction and lenalidomide in maintenance), and the vincristine, doxorubicin, and dexamethasone (VAD) group of the HOVON65/GMMG-HD4 trial (thalidomide in maintenance). The primary endpoint was to show the prognostic value of the IMiD-14 gene signature for progression-free survival. FINDINGS: In the training cohort, progression-free survival was significantly shorter in the 83 patients with IMiD-14 high scores than in the 92 patients with IMiD-14 low scores; 3 year progression-free survival was 52% (95% CI 42-64) for the IMiD-14 high group versus 85% (78-92) for the IMiD-14 low group, with a hazard ratio (HR) of 2·51 (95% CI 1·72-3·66; p<0·0001). These findings were supported by the results in the validation cohorts, TT3a (115 patients with IMiD-14 high vs 160 patients with IMiD-14 low; 3 year progression-free survival 63% [95% CI 55-73] vs 87% [82-92]; HR 1·54 [1·11-2·15], p=0·010), TT3b (77 patients with IMiD-14 high vs 89 patients with IMiD-14 low; 62% [52-74] vs 80% [72-89]; HR 2·07 [1·28-3·34], p=0·0024), TT6 (20 patients with IMiD-14 high vs 36 patients with IMiD-14 low; 39% [22-68] vs 74% [61-90]; HR 2·40 [1·09-5·30], p=0·026), and the VAD group of HOVON65/GMMG-HD4 (65 patients with IMiD-14 high vs 77 patients with IMiD-14 low; 16% [9-28] vs 54% [44-67]; HR 2·29 [1·52-3·45], p<0·0001). INTERPRETATION: Our results suggest that the IMiD-14 model has prognostic value in patients with multiple myeloma who are treated with IMiDs. Some genes in the model could provide novel targets for IMiD resistance and therapeutic intervention. The IMiD-14 model warrants evaluation in prospective studies. FUNDING: Conquer Cancer Foundation ASCO Young Investigator Award and the Carolinas Myeloma Research Fund.

Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis.

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFß1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1.

Use of molecular markers to determine postremission treatment in acute myeloid leukemia with normal cytogenetics.

Most patients with acute myeloid leukemia can be induced into complete remission, but postremission treatment is required for cure. The choice of postremission therapy in a majority of nonelderly patients, between intensive chemotherapy and allogeneic hematopoietic cell transplantation, is largely determined by the results of conventional cytogenetic analysis. In 45% of patients with a normal karyotype, the presence or absence of specific molecular mutations should be used to determine the prognosis and postremission treatment. In addition, the identification of mutations may indicate a role for targeted intervention, including following transplantation.

Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

Heat shock factor-1 knockout induces multidrug resistance gene, MDR1b, and enhances P-glycoprotein (ABCB1)-based drug extrusion in the heart.

Heat-shock factor 1 (HSF-1), a transcription factor for heat-shock proteins (HSPs), is known to interfere with the transcriptional activity of many oncogenic factors. In the present work, we have discovered that HSF-1 ablation induced the multidrug resistance gene, MDR1b, in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug-resistant cancer cells. The increase in P-gp enhanced the extrusion of doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Dox-induced left ventricular (LV) dysfunction was significantly reduced in HSF-1(-/-) mice. DNA-binding activity of NF-κB was higher in HSF-1(-/-) mice. IκB, the NF-κB inhibitor, was depleted due to enhanced IκB kinase (IKK)-α activity. In parallel, MDR1b gene expression and a large increase in P-gp and lowering Dox loading were observed in HSF-1(-/-) mouse hearts. Moreover, application of the P-gp antagonist, verapamil, increased Dox loading in HSF-1(-/-) cardiomyocytes, deteriorated cardiac function in HSF-1(-/-) mice, and decreased survival. MDR1 promoter activity was higher in HSF-1(-/-) cardiomyocytes, whereas a mutant MDR1 promoter with heat-shock element (HSE) mutation showed increased activity only in HSF-1(+/+) cardiomyocytes. However, deletion of HSE and NF-κB binding sites diminished luminescence in both HSF-1(+/+) and HSF-1(-/-) cardiomyocytes, suggesting that HSF-1 inhibits MDR1 activity in the heart. Thus, because high levels of HSF-1 are attributed to poor prognosis of cancer, systemic down-regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapy-induced cardiotoxicity and enhance cancer prognosis.

Heat shock protein 25-enriched plasma transfusion preconditions the heart against doxorubicin-induced dilated cardiomyopathy in mice.

Extracellular heat shock proteins (eHsps) in the circulation have recently been found to activate both apoptotic and protective signaling in the heart. However, the role of eHsps in doxorubicin (Dox)-induced heart failure has not yet been studied. The objective of the present study was to determine how Dox affects circulating eHsp25 in blood plasma and how eHsp25 affects Dox-induced dilated cardiomyopathy. Wild-type mice [HSF-1(+/+)] were pretreated with 100 µl of heterozygous heat shock factor-1 [HSF-1(+/-)] mouse plasma (which contained 4-fold higher eHsp25 compared with wild-type mice), HSF-1(+/+) plasma, or saline, before treatment with Dox (6 mg/kg). After 4 weeks of this treatment protocol, HSF-1(+/-) plasma-pretreated mice showed increased eHsp25 in plasma and improved cardiac function (percentage of fractional shortening 37.3 ± 2.1 versus 26.4 ± 4.0) and better life span (31 ± 2 versus 22 ± 3 days) compared with the HSF-1(+/+) plasma or saline-pretreated mice. Preincubation of isolated adult cardiomyocytes with HSF-1(+/-) plasma or recombinant human Hsp27 (rhHsp27) significantly reduced Dox-induced activation of nuclear factor-κB and cytokine release and delayed cardiomyocyte death. Moreover, when cardiomyocytes were incubated with fluorescence-tagged rhHsp27, a saturation in binding was observed, suggesting that eHsp25 can bind to surface receptors. Competitive assays with a Toll-like receptor 2 (TLR2) antibody reduced the rhHSP27 binding, indicating that Hsp25 interacts with TLR2. In conclusion, transfusion of Hsp25-enriched blood plasma protected the heart from Dox-induced cardiotoxicity. Hsp25 antagonized Dox binding to the TLR2 receptor on cardiomyocytes.