Melanie E. Davis

The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

Phase II study of the histone deacetylase inhibitor MGCD0103 in patients with previously treated chronic lymphocytic leukaemia

MGCD0103, an orally available class I histone deacetylase (HDAC) inhibitor, was examined for pre‐clinical activity in chronic lymphocytic leukaemia (CLL). A phase II clinical trial was performed, starting at a dose of 85 mg/d, three times per week. Dose escalation to 110 mg or the addition of rituximab was permitted in patients without a response after two or more cycles. MGCD0103 demonstrated pre‐clinical activity against CLL cells with a LC50 (concentration lethal to 50%) of 0·23 μmol/l and increased acetylation of the HDAC class I specific target histone H3. Twenty‐one patients received a median of two cycles of MGCD0103 (range, 0–12). All patients had previously received fludarabine, 33% were fludarabine refractory, and 71% had del(11q22·3) or del(17p13·1). No responses according to the National Cancer Institutes 1996 criteria were observed. Three patients received 110 mg and four patients received concomitant rituximab, with no improvement in response. Grade 3–4 toxicity consisted of infections, thrombocytopenia, anaemia, diarrhoea, and fatigue. HDAC inhibition was observed in six out of nine patients on day 8. Limited activity was observed with single agent MGCD0103 in high risk patients with CLL. Future investigations in CLL should focus on broad HDAC inhibition, combination strategies, and approaches to diminish constitutional symptoms associated with this class of drugs.

Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Brutons tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Mcl-1 expression predicts progression-free survival in chronic lymphocytic leukemia patients treated with pentostatin, cyclophosphamide, and rituximab

Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. Increased Mcl-1 expression is associated with failure to achieve remission after treatment with fludarabine and chlorambucil in patients with chronic lymphocytic leukemia (CLL). However, the influence of Mcl-1 expression has not been examined in CLL trials using chemoimmunotherapy. We investigated Mcl-1 protein expression prospectively as part of a phase 2 study evaluating the efficacy of pentostatin, cyclophosphamide, and rituximab in patients with untreated CLL. No significant difference by Mcl-1 expression was noted in pretreatment or response parameters. However, in patients with higher Mcl-1 expression, both minimal residual disease-negative status and progression-free survival was found to be significantly reduced (57% vs 19%, P = .01; 50.8 vs 18.7 months; P = .02; respectively). Mcl-1 expression may therefore be useful in predicting poor response to chemoimmunotherapy. These findings further support pursuing treatment strategies targeting this important antiapoptotic protein. (Because the trials described were conducted before the requirement to register them was implemented, they are not registered in a clinical trial database.).

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

Background While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies. Principal Findings In mantle cell lymphoma (JeKo-1), Burkitts lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC50 (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC50 (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity. Conclusions/Significance Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.

BTKC481S-Mediated Resistance to Ibrutinib in Chronic Lymphocytic Leukemia.

Purpose Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and Methods Patients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. Results With a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. Conclusion Relapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.

Resistance to the Translation Initiation Inhibitor Silvestrol is Mediated by ABCB1/P-Glycoprotein Overexpression in Acute Lymphoblastic Leukemia Cells

Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.

The Proteasome Inhibitor Carfilzomib Functions Independently of p53 to Induce Cytotoxicity and an Atypical NF-κB Response in Chronic Lymphocytic Leukemia Cells

Purpose: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. Experimental Design: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line. Results: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. Conclusions: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease. Clin Cancer Res; 19(9); 2406–19. ©2013 AACR.

Validation of an LC-MS Based Approach for Profiling Histones in Chronic Lymphocytic Leukemia

The in vitro evaluation of histones and their PTMs has drawn substantial interest in the development of epigenetic therapies. The differential expression of histone isoforms may serve as a potential marker in the classification of diseases affected by chromatin abnormalities. In this study, protein profiling by LC and MS was used to explore differences in histone composition in primary chronic lymphocytic leukemia (CLL) cells. Extensive method validations were performed to determine the experimental variances that would impact histone relative abundance. The resulting data demonstrated that the proposed methodology was suitable for the analysis of histone profiles. In 4 normal individuals and 40 CLL patients, a significant decrease in the relative abundance of histone H2A variants (H2AFL and H2AFA/M*) was observed in primary CLL cells as compared to normal B cells. Protein identities were determined using high mass accuracy MS and shotgun proteomics.

A Phase I Trial to Evaluate Antibody-Dependent Cellular Cytotoxicity of Cetuximab and Lenalidomide in Advanced Colorectal and Head and Neck Cancer

mAbs can induce antibody-dependent cellular cytotoxicity (ADCC) via the innate immune systems ability to recognize mAb-coated cancer cells and activate immune effector cells. Lenalidomide is an immunomodulatory agent with the capacity to stimulate immune cell cytokine production and ADCC activity. This phase I trial evaluated the combination of cetuximab with lenalidomide for the treatment of advanced colorectal and head and neck squamous cell cancers (HNSCC). This trial included patients with advanced colorectal cancer or HNSCC. Treatment consisted of cetuximab 500 mg/m2 i.v. every two weeks with lenalidomide given orally days 1–21 on a 28-day cycle. Three dose levels of lenalidomide were evaluated (15, 20, 25 mg). Correlative studies included measurement of ADCC, FcγRIIIA polymorphism genotyping, measurement of serum cytokine levels, and flow cytometric analysis of immune cell subtypes. Twenty-two patients were enrolled (19 colorectal cancer, 3 HNSCC). Fatigue was the only dose-limiting toxicity. One partial response was observed and 8 patients had stable disease at least 12 weeks. The recommended phase II dose is cetuximab 500 mg/m2 with lenalidomide 25 mg daily, days 1–21. Correlative studies demonstrated a dose-dependent increase in natural killer cytotoxic activity with increasing doses of lenalidomide. Cetuximab and lenalidomide were well tolerated. There was a lenalidomide dose-dependent increase in ADCC with higher activity in patients enrolled in cohort 3 than those enrolled in cohorts 1/2. Although response was not a primary endpoint, there was evidence of antitumor activity for the combination therapy. Further investigation of lenalidomide as an immunomodulator in solid tumors is warranted. Mol Cancer Ther; 15(9); 2244–50. ©2016 AACR.