ALBERT

Description: Journal of Medicinal Chemistry DOI: 10.1021/jm301367c

Description: Background: Mutations of MAP2K1, which encodes MEK1, have been identified in up to half of patients with variant Hairy Cell Leukemia (vHCL).[Waterfall et al., Nat Gen 2014, Mason et al., Leukemia & Lymphoma 2016], and have been associated with vHCL with IGHV4-34 gene usage, which This form of HCL tends to have a worse prognosis than classic HCL or wild type vHCL (Arons et al., Blood 2009), with inferior responses to chemotherapy and shorter durations of remission. Trametinib, an oral inhibitor of MEK1 and MEK2, is FDA approved for treatment of patients with BRAF p.V600E mutant melanoma. We hypothesized that this MEK inhibitor would have activity in MAP2K1 mutant vHCL. Case Report: The patient is a 52 year old man with a history of CD25+, BRAF wildtype, IGHV4-34 usage vHCL diagnosed in 2005. His previous treatments included cladribine, BL22, pentostatin/rituximab, splenectomy, single agent rituximab, ibrutinib, bendamustine/rituximab, and allogeneic transplantation from a matched unrelated donor. The patient experienced disease relapse day +350 post transplant when he developed skin nodules as well as a generalized skin rash. The skin rash appeared clinically consistent with acute GVHD. However, when biopsies of both the skin nodules and skin rash were performed he was found to have relapsed vHCL. He was consented for paired tumor and germline next generation sequencing with a 25-gene amplicon panel which revealed a somatic MAP2K1 K57N mutation that has been shown to constitutively activate MEK [Marks et al., Cancer Res 2008]. As the patient had exhausted the majority of available treatment options, he was prescribed trametinib 2 mg po daily (commercial supply, according to approved melanoma dosing). Within a week of therapy initiation his skin nodules were markedly diminished in size and his generalized rash had resolved. He did develop a new acneiform rash over his face consistent with drug toxicity. This was managed with topical agents with improvement and did not require a dose reduction. Disease restaging following cycle 2 of therapy showed near complete resolution of skin nodules, with disappearance of visible skin rash. Repeat bone marrow biopsy showed unchanged hairy cell index. Skin biopsies were repeated and phospho-ERK (T202/Y204) staining of skin biopsies pre- and post-trametinib were performed (Figure 1). This showed diminished lymphocyte involvement on H&E staining with a decrease in p-ERK expression on immunostaining, indicative of decreased signaling downstream of MEK and consistent with on target trametinib effects. As of this writing, the patient has remained on trametinib for 12 weeks with no recurrence of leukemia cutis rash. Discussion: MEK inhibition with the oral MEKi trametinib is a well tolerated therapy with clinical activity in MAP2K1 mutant vHCL. Additional studies of this agent are warranted. Optimal dose and duration of therapy will need to be explored in prospective clinical trials. Figure 1 Skin biopsies pre- and post-trametinib. (A)(C) H&E staining shows diminished lymphocyte involvement. (B)(D) PhosphoERK immunostaining shows decrease of phosphoERK expression. Bar = 500 μm Figure 1. Skin biopsies pre- and post-trametinib. (A)(C) H&E staining shows diminished lymphocyte involvement. (B)(D) PhosphoERK immunostaining shows decrease of phosphoERK expression. Bar = 500 μm Disclosures Andritsos: Hairy Cell Leukemia Foundation: Research Funding. Anghelina:Hairy Cell Leukemia Foundation: Research Funding. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding. Jones:Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Restoring nuclear localization of tumor suppressors by blocking exportin 1 (XPO1) holds promise as a new therapeutic paradigm in many cancers, including chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Oral selective inhibitor of nuclear export (SINE) compounds that covalently modify XPO1 were recently discovered and are exciting new compounds to implement this strategy. Selinexor, the clinical lead SINE, has made progress in Phase I/II clinical trials and is generally well tolerated, but limited to twice weekly dosing, with supportive care. The discovery of novel SINE compounds with improved tolerability is therefore of considerable clinical interest and represents a significant contribution beyond the targeted therapies currently available for hematologic malignancies and a variety of other cancers where upregulation of XPO1 is observed. Presented herein is the discovery of KPT-8602, the next generation SINE compound that shows lower brain penetration, improved tolerability allowing continuous dosing, and improved efficacy beyond any current XPO1 inhibitor. Results: Our crystallography data revealed that KPT-8602 binds covalently to XPO1 through a Michael acceptor that is activated by an electron withdrawing pyrimidyl moiety, allowing a 2-fold increased reversible interaction with XPO1 compared to earlier SINE compounds. The crystal structure of the KPT-8602-XPO1 complex showed interactions between XPO1 and the activating pyrimidyl group. In vitro pull-downs using immobilized GST-nuclear export sequences and purified recombinant XPO1 demonstrated greater reversible binding of KPT-8602 compared to KPT-330 (selinexor). In vivo toxicology studies demonstrated that KPT-8602 possesses lower brain penetration compared to KPT-330 allowing for continuous dosing and improved tolerability. Our results also showed that KPT-8602 induced a comparable level of cytotoxicity as well as inhibition of cell proliferation compared to KPT-330 in primary CLL tumors and in a representative panel of DLBCL cell lines. Furthermore, KPT-8602 inhibited proliferation and induced apoptosis in AML cell lines and primary AML blasts while inducing nuclear accumulation of p53 and NPM1. We hypothesized that these improved pharmacological parameters would allow daily KPT-8602 to abrogate disease progression in CLL and AML animal models. The Eµ-TCL1-C57BL/6 transplant model of CLL was used to evaluate the therapeutic benefit of continuous dosing of KPT-8602. Eµ-TCL1-engrafted mice were treated with KPT-8602 given daily or 2x/week. The KPT-8602 daily cohort had significantly improved survival with a median overall survival of 70 vs 50 days (vs vehicle 33 days), compared to those treated only 2x/week with KPT-8602 (p=0.001). Mice treated with KPT-330 2x/week showed a similar survival to mice treated with KPT-8602 2x/week. Mice given daily KPT-8602 had significantly smaller spleens and reduced circulating leukemic cells compared to all the other groups (p

Description: Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 〈 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 886 Patients with chronic lymphocytic leukemia (CLL) having del(17p13.1), complex karyotype, or fludarabine-refractory disease have few therapeutic options. The cyclin-dependent kinase (CDK) inhibitor flavopiridol (Alvocidib) has been demonstrated to be very effective in genetically high-risk and fludarabine-refractory CLL cases, with a response rate of approximately 50%. We pursued pre-clinical and now early clinical studies of SCH 727965. SCH 727965 is a selective inhibitor of CDK 1, 2, 5 and 9 (IC50 of 〈 5nM) that shows in vitro and in vivo anti-tumor activity in a variety of pre-clinical models. Notably, SCH 727965 has an improved therapeutic index over other CDK inhibitors such as flavopiridol. A solid tumor phase I study with SCH 727965 administered by 2-hour IV infusion on days 1, 8 and 15 of a 28-day cycle has been completed, resulting in a recommended phase II dose of 12 mg/m2. Our pre-clinical studies with SCH 727965 demonstrate potent induction of apoptosis in CLL patient cells following a 2-hour incubation, with an LC50 of 240 nM. This effect is independent of prior treatment status, IgVH gene mutational status, and high-risk cytogenetic abnormalities. Importantly, minimal cytotoxicity toward normal T cells is observed. Similar to other CDK inhibitors in CLL, SCH 727965 promotes rapid down-regulation of the pro-survival gene MCL1 at both the mRNA and protein levels. Stimulation of CLL cells with CD40L mimics the lymph node microenvironment and increases resistance of CLL cells to drug-induced apoptosis (Smit et al., Blood 109:1660-68, 2007). However, co-treatment of CLL cells with SCH 727965 plus CD40L does not abrogate the apoptotic effect of SCH 727965. Based upon these promising data, a disease-specific phase I study of SCH 727965 has been initiated in previously treated CLL patients. This trial started at a dose level below the phase II dose established in the solid tumor study, due to the previous observation of hyperacute tumor lysis in CLL with other CDK inhibitors. SCH 727965 is administered by 2-hour IV infusion on days 1, 8 and 15 of a 28-day cycle. Dosing cohorts are: 5 mg/m2, 7 mg/m2, 10 mg/m2, and 14 mg/m2. Further dose-escalation is allowed, depending on the frequency of dose limiting toxicities observed. To date, we have completed enrollment to cohorts 1 and 2 of the study, with ten patients enrolled. Clinical features of the patients include a median age of 58 (range 43 to 73), 60% Rai stage 3 or 4, and 60% with high-risk genomic features (del17p13, del11q22, or more than two abnormalities). These patients received a median of six (range 1 to 12) prior therapies, and 90% received prior fludarabine therapy. Biochemical evidence of tumor lysis (increased potassium, phosphate, or LDH) post-treatment has been observed in three patients. Common toxicities observed include: nausea, vomiting, diarrhea and fatigue. One patient with pre-treatment neutropenia developed fatal bacterial sepsis during cycle 1 of therapy. Three patients have discontinued therapy due to progressive disease after cycle one, including one patient who died from CLL disease. Two patients discontinued therapy with stable disease. Four patients remain on therapy; one in the lowest dose cohort has demonstrated a partial response by IWCLL 2008 criteria. The other three patients currently are early in the course of planned therapy. The clinical observation of rapid improvement in palpable adenopathy after SCH 727965 treatment is consistent with the laboratory observation of continued sensitivity of CD40L-exposed CLL cells to SCH 727965. Pharmacodynamic studies demonstrate down-modulation of MCL1 in a subset of patients. These data provide promising pre-clinical, and now early clinical, evidence of the potential therapeutic benefit of SCH 727965 in CLL. Dose escalation of SCH 727965 continues and updated results will be presented. This work was supported by the D. Warren Brown Foundation and Specialized Center of Research from the Leukemia and Lymphoma Society. Disclosures: Small: Schering Plough: Employment. Statkevich:Schering Plough: Employment. Bannerji:Schering-Plough: Employment.

Description: Initial therapy for Chronic Lymphocytic Leukemia (CLL) typically includes nucleoside analogs such as fludarabine. Mutations of p53 are uncommon in CLL patients at diagnosis, but are found at increased frequency in patients with prior treatment and are often associated with resistance to standard therapies including fludarabine. The causality of fludarabine treatment to p53 mutation has not been clearly defined. In previous work with the TCL-1 model of CLL, we demonstrated that mice with active leukemia do not have p53 mutations and respond to fludarabine therapy with a modest survival advantage (Johnson et al, Blood108:1334, 2006). We therefore initiated a randomized trial of fludarabine treatment (34mg/kg daily intraperitoneal injection for five days every 28 days) versus observation (treatment with volume-matched vehicle controls on the same dosing schedule) in TCL-1 transgenic mice prior to the onset of leukemia. The goals of this study were to determine if early treatment with fludarabine would 1) delay disease onset; 2) eventually yield a fludarabine-resistant phenotype; or 3) promote development of p53 mutations in mice developing progressive leukemia. Transgenic TCL-1 mice between the ages of eight and 12 weeks were continually treated with fludarabine (n=32) or saline (n=45) until death while being screened monthly for disease progression through changes in WBC count by peripheral blood differentiation smear, lymphocyte subsets by flow cytometry, and nodal and spleen exams. Despite continued treatment with fludarabine, drug resistance as manifested by leukemia and eventual death occurred. Median time to leukemia onset was 308 days (95% CI: 224, ∞) in the fludarabine group and 338 days (95% CI: 252, ∞) in the saline group. Thus, the risk of developing leukemia was not significantly different between the two groups. Of interest, p53 mutations in exons 2–11 were not found in either group (n=10 each) at the time of death. Microarray studies are currently underway to discover other genes differentially expressed between treated and untreated mice that might contribute to fludarabine resistance and will be presented. In conclusion, our data demonstrate that early fludarabine treatment in the TCL-1 transgenic model of CLL does not prevent onset of leukemia, but instead results in disease resistant to fludarabine. The source of fludarabine resistance in this mouse model was not through acquisition of p53 mutations, but rather an alternative unidentified mechanism(s).

Description: Despite promising preclinical studies with the cyclin-dependent kinase inhibitor flavopiridol in chronic lymphocytic leukemia (CLL) and other diseases, previous clinical trials with this agent have been disappointing. The discovery of differential protein binding of flavopiridol in human and bovine serum contributed to an effective pharmacokinetic-derived schedule of administration of this agent. On the basis of pharmacokinetic modeling using our in vitro results and data from a previous trial, we initiated a phase 1 study using a 30-minute loading dose followed by 4 hours of infusion administered weekly for 4 of 6 weeks in patients with refractory CLL. A group of 42 patients were enrolled on 3 cohorts (cohort 1, 30 mg/m2 loading dose followed by 30 mg/m2 4-hour infusion; cohort 2, 40 mg/m2 loading dose followed by 40 mg/m2 4-hour infusion; and cohort 3, cohort 1 dose for treatments 1 to 4, then a 30 mg/m2 loading dose followed by a 50 mg/m2 4-hour infusion). The dose-limiting toxicity using this novel schedule was hyperacute tumor lysis syndrome. Aggressive prophylaxis and exclusion of patients with leukocyte counts greater than 200 × 109/L have made this drug safe to administer at the cohort 3 dose. Of the 42 patients treated, 19 (45%) achieved a partial response with a median response duration that exceeds 12 months. Responses were noted in patients with genetically high-risk disease, including 5 (42%) of 12 patients with del(17p13.1) and 13 (72%) of 18 patients with del(11q22.3). Flavopiridol administered using this novel schedule has significant clinical activity in refractory CLL. Patients with bulky disease and high-risk genetic features have achieved durable responses, thereby justifying further study of flavopiridol in CLL and other diseases.