Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML.

Post-transplantation B cell activating factor and B cell recovery before onset of chronic graft-versus-host disease.

Excessive levels of B cell activating factor (BAFF) are found in patients with active chronic graft-versus-host disease (cGVHD). In mice, BAFF has been shown to be essential for B cell recovery after myeloablation. To assess how BAFF levels relate to transplantation factors and subsequent development of cGVHD, we prospectively monitored 412 patients in the first year after allogeneic peripheral blood or bone marrow hematopoietic stem cell transplantation (HSCT) and censored data at time of cGVHD onset. In patients who did not develop cGVHD, we affirmed a temporal pattern of gradually decreasing BAFF levels as B cell numbers increase after myeloablative conditioning. In contrast, after reduced-intensity conditioning, BAFF levels remained high throughout the first post-HSCT year, suggesting that the degree of myeloablation resulted in delayed B cell recovery associated with persistence of higher BAFF levels. Given that high BAFF/B cell ratios have been associated with active cGVHD, we examined differences in early BAFF/B cell ratios and found significantly different BAFF/B cell ratios at 3 months post-HSCT only after myeloablative conditioning in patients who subsequently developed cGVHD. In addition to HSCT conditioning type, the use of sirolimus was significantly associated with higher BAFF levels after HSCT, and this also was potentially related to lower B cell numbers. Taken together, our results are important for interpreting BAFF measurements in cGVHD biomarker studies.

Autologous CLL cell vaccination early after transplant induces leukemia-specific T cells.

BACKGROUND: Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. METHODS: We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. RESULTS: At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. CONCLUSION: Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. TRIAL REGISTRATION: Clinicaltrials.gov NCT00442130. FUNDING: NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Bortezomib-based graft-versus-host disease prophylaxis in HLA-mismatched unrelated donor transplantation.

PURPOSE: HLA-mismatched unrelated donor (MMUD) hematopoietic stem-cell transplantation (HSCT) is associated with increased graft-versus-host disease (GVHD) and impaired survival. In reduced-intensity conditioning (RIC), neither ex vivo nor in vivo T-cell depletion (eg, antithymocyte globulin) convincingly improved outcomes. The proteasome inhibitor bortezomib has immunomodulatory properties potentially beneficial for control of GVHD in T-cell-replete HLA-mismatched transplantation. PATIENTS AND METHODS: We conducted a prospective phase I/II trial of a GVHD prophylaxis regimen of short-course bortezomib, administered once per day on days +1, +4, and +7 after peripheral blood stem-cell infusion plus standard tacrolimus and methotrexate in patients with hematologic malignancies undergoing MMUD RIC HSCT. We report outcomes for 45 study patients: 40 (89%) 1-locus and five (11%) 2-loci mismatches (HLA-A, -B, -C, -DRB1, or -DQB1), with a median of 36.5 months (range, 17.4 to 59.6 months) follow-up. RESULTS: The 180-day cumulative incidence of grade 2 to 4 acute GVHD was 22% (95% CI, 11% to 35%). One-year cumulative incidence of chronic GVHD was 29% (95% CI, 16% to 43%). Two-year cumulative incidences of nonrelapse mortality (NRM) and relapse were 11% (95% CI, 4% to 22%) and 38% (95% CI, 24% to 52%), respectively. Two-year progression-free survival and overall survival were 51% (95% CI, 36% to 64%) and 64% (95% CI, 49% to 76%), respectively. Bortezomib-treated HLA-mismatched patients experienced rates of NRM, acute and chronic GVHD, and survival similar to those of contemporaneous HLA-matched RIC HSCT at our institution. Immune recovery, including CD8(+) T-cell and natural killer cell reconstitution, was enhanced with bortezomib. CONCLUSION: A novel short-course, bortezomib-based GVHD regimen can abrogate the survival impairment of MMUD RIC HSCT, can enhance early immune reconstitution, and appears to be suitable for prospective randomized evaluation.

Immune reconstitution after double umbilical cord blood stem cell transplantation: comparison with unrelated peripheral blood stem cell transplantation.

Double umbilical cord blood (DUCB) transplantation is an accepted transplantation strategy for patients without suitable human leukocyte antigen (HLA) matched donors. However, DUCB transplantation is associated with increased morbidity and mortality because of slow recovery of immunity and a high risk of infection. To define the differences in immune reconstitution between DUCB transplantation and HLA matched unrelated donor (MUD) transplantation, we performed a detailed, prospective analysis of immune reconstitution in 42 DUCB recipients and 102 filgrastim-mobilized unrelated peripheral blood stem cell recipients. Reconstitution of CD3 T cells was significantly delayed in the DUCB cohort compared with the MUD cohort for 1 to 6 months posttransplantation (P < .001), including naive (CD45RO-) and memory (CD45RO+) CD4 T cells, regulatory (CD4CD25) T cells, and CD8 T cells. In contrast, CD19 B cells recovered more rapidly in the DUCB cohort and numbers remained significantly greater from 3 to 24 months after transplantation (P = .001). CD56CD16 natural killer (NK) cells also recovered more rapidly in DUCB recipients and remained significantly greater from 1 to 24 months after transplantation. B cell activating factor (BAFF) levels were higher in the DUCB cohort at 1 month (P < .001), were similar in both cohorts at 3 and 6 months, and were lower in the DUCB cohort at 12 months (P = .002). BAFF/CD19 B cell ratios were lower in the DUCB cohort at 3 (P = .045), 6 (P = .02), and 12 months (P = .002) after transplantation. DUCB recipients had more infections within the first 100 days after transplantation (P < .001), and there was less chronic graft-versus-host disease (P < .001), but there were no differences in cumulative incidence of relapse, nonrelapse death, progression-free survival, or overall survival between the 2 groups. These results suggest that increased risk of infections is specifically associated with delayed reconstitution of all major T cell subsets, but the increased risk is limited to the first 3 months after DUCB transplantation. There is no increased risk of relapse, suggesting that graft-versus-leukemia activity is maintained. Early reconstitution of B cells and NK cells may, in part, account for these findings.

Interleukin-2 and regulatory T cells in graft-versus-host disease.

BACKGROUND: Dysfunction of regulatory T (Treg) cells has been detected in diverse inflammatory disorders, including chronic graft-versus-host disease (GVHD). Interleukin-2 is critical for Treg cell growth, survival, and activity. We hypothesized that low-dose interleukin-2 could preferentially enhance Treg cells in vivo and suppress clinical manifestations of chronic GVHD. METHODS: In this observational cohort study, patients with chronic GVHD that was refractory to glucocorticoid therapy received daily low-dose subcutaneous interleukin-2 (0.3×10(6), 1×10(6), or 3×10(6) IU per square meter of body-surface area) for 8 weeks. The end points were safety and clinical and immunologic response. After a 4-week hiatus, patients with a response could receive interleukin-2 for an extended period. RESULTS: A total of 29 patients were enrolled. None had progression of chronic GVHD or relapse of a hematologic cancer. The maximum tolerated dose of interleukin-2 was 1×10(6) IU per square meter. The highest dose level induced unacceptable constitutional symptoms. Of the 23 patients who could be evaluated for response, 12 had major responses involving multiple sites. The numbers of CD4+ Treg cells were preferentially increased in all patients, with a peak median value, at 4 weeks, that was more than eight times the baseline value (P<0.001), without affecting CD4+ conventional T (Tcon) cells. The Treg:Tcon ratio increased to a median of more than five times the baseline value (P<0.001). The Treg cell count and Treg:Tcon ratio remained elevated at 8 weeks (P<0.001 for both comparisons with baseline values), then declined when the patients were not receiving interleukin-2. The increased numbers of Treg cells expressed the transcription factor forkhead box P3 (FOXP3) and could inhibit autologous Tcon cells. Immunologic and clinical responses were sustained in patients who received interleukin-2 for an extended period, permitting the glucocorticoid dose to be tapered by a mean of 60% (range, 25 to 100). CONCLUSIONS: Daily low-dose interleukin-2 was safely administered in patients with active chronic GVHD that was refractory to glucocorticoid therapy. Administration was associated with preferential, sustained Treg cell expansion in vivo and amelioration of the manifestations of chronic GVHD in a substantial proportion of patients. (Funded by a Dana-Farber Dunkin' Donuts Rising Star award and others; ClinicalTrials.gov number, NCT00529035.).

PD-1 on immature and PD-1 ligands on migratory human Langerhans cells regulate antigen-presenting cell activity.

Langerhans cells (LCs) are known as "sentinels" of the immune system that function as professional antigen-presenting cells (APCs) after migration to draining lymph node. LCs are proposed to have a role in tolerance and the resolution of cutaneous immune responses. The Programmed Death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, are a co-inhibitory pathway that contributes to the negative regulation of T-lymphocyte activation and peripheral tolerance. Surprisingly, we found PD-1 to be expressed on immature LCs (iLCs) in situ. PD-1 engagement on iLCs reduced IL-6 and macrophage inflammatory protein (MIP)-1alpha cytokine production in response to TLR2 signals but had no effect on LC maturation. PD-L1 and PD-L2 were expressed at very low levels on iLCs. Maturation of LCs upon migration from epidermis led to loss of PD-l expression and gain of high expression of PD-L1 and PD-L2 as well as co-stimulatory molecules. Blockade of PD-L1 and/or PD-L2 on migratory LCs (mLCs) and DDCs enhanced T-cell activation, as has been reported for other APCs. Thus the PD-1 pathway is active in iLCs and inhibits iLC activities, but expression of receptor and ligands reverses upon maturation and PD-L1 and PD-L2 on mLC function to inhibit T-cell responses.