Jason Claud Chandler

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNAs seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

Aberrant Overexpression of IL-15 Initiates Large Granular Lymphocyte Leukemia through Chromosomal Instability and DNA Hypermethylation

How inflammation causes cancer is unclear. Interleukin-15 (IL-15) is a pro-inflammatory cytokine elevated in human large granular lymphocyte (LGL) leukemia. Mice overexpressing IL-15 develop LGL leukemia. Here, we show that prolonged in vitro exposure of wild-type (WT) LGL to IL-15 results in Myc-mediated upregulation of aurora kinases, centrosome aberrancies, and aneuploidy. Simultaneously, IL-15 represses miR-29b via induction of Myc/NF-κBp65/Hdac-1, resulting in Dnmt3b overexpression and DNA hypermethylation. All this is validated in human LGL leukemia. Adoptive transfer of WT LGL cultured with IL-15 led to malignant transformation in vivo. Drug targeting that reverses miR-29b repression cures otherwise fatal LGL leukemia. We show how excessive IL-15 initiates cancer and demonstrate effective drug targeting for potential therapy of human LGL leukemia.

Dose Escalation of Lenalidomide in Relapsed or Refractory Acute Leukemias

PURPOSE Lenalidomide is effective in myeloma and low-risk myelodysplastic syndromes with deletion 5q. We report results of a phase I dose-escalation trial of lenalidomide in relapsed or refractory acute leukemia. PATIENTS AND METHODS Thirty-one adults with acute myeloid leukemia (AML) and four adults with acute lymphoblastic leukemia (ALL) were enrolled. Lenalidomide was given orally at escalating doses of 25 to 75 mg daily on days 1 through 21 of 28-day cycles to determine the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD), as well as to provide pharmacokinetic and preliminary efficacy data. RESULTS Patients had a median age of 63 years (range, 22 to 79 years) and a median of two prior therapies (range, one to four therapies). The DLT was fatigue; 50 mg/d was the MTD. Infectious complications were frequent. Plasma lenalidomide concentration increased proportionally with dose. In AML, five (16%) of 31 patients achieved complete remission (CR); three of three patients with cytogenetic abnormalities achieved cytogenetic CR (none with deletion 5q). Response duration ranged from 5.6 to 14 months. All responses occurred in AML with low presenting WBC count. No patient with ALL responded. Two of four patients who received lenalidomide as initial therapy for AML relapse after allogeneic transplantation achieved durable CR after development of cutaneous graft-versus-host disease, without donor leukocyte infusion. CONCLUSION Lenalidomide was safely escalated to 50 mg daily for 21 days, every 4 weeks, and was active with relatively low toxicity in patients with relapsed/refractory AML. Remissions achieved after transplantation suggest a possible immunomodulatory effect of lenalidomide, and results provide enthusiasm for further studies in AML, either alone or in combination with conventional agents or other immunotherapies.

Avelumab for patients with previously treated metastatic or recurrent non-small-cell lung cancer (JAVELIN Solid Tumor): dose-expansion cohort of a multicentre, open-label, phase 1b trial

BACKGROUND Avelumab, a human Ig-G1 monoclonal antibody targeting PD-L1 and approved in the USA for the treatment of metastatic Merkel cell carcinoma, has shown antitumour activity and an acceptable safety profile in patients with advanced solid tumours in a dose-escalation phase 1a trial. In this dose-expansion cohort of that trial, we assess avelumab treatment in a cohort of patients with advanced, platinum-treated non-small-cell lung cancer (NSCLC). METHODS In this dose-expansion cohort of a multicentre, open-label, phase 1 study, patients with progressive or platinum-resistant metastatic or recurrent NSCLC were enrolled at 58 cancer treatment centres and academic hospitals in the USA. Eligible patients had confirmed stage IIIB or IV NSCLC with squamous or non-squamous histology, measurable disease by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1), tumour biopsy or archival sample for biomarker assessment, and Eastern Cooperative Oncology Group performance status 0 or 1, among other criteria. Patient selection was not based on PD-L1 expression or expression of other biomarkers, including EGFR or KRAS mutation or ALK translocation status. Patients received infusional avelumab monotherapy 10 mg/kg every 2 weeks until disease progression or toxicity. The primary objective was to assess safety and tolerability. This trial is registered with ClinicalTrials.gov, number NCT01772004; enrolment in this cohort is closed and the trial is ongoing. FINDINGS Between Sept 10, 2013, and June 24, 2014, 184 patients were enrolled and initiated treatment with avelumab. Median follow-up duration was 8·8 months (IQR 7·2-11·9). The most common treatment-related adverse events of any grade were fatigue (46 [25%] of 184 patients), infusion-related reaction (38 [21%]), and nausea (23 [13%]). Grade 3 or worse treatment-related adverse events occurred in 23 (13%) of 184 patients; the most common (occurring in more than two patients) were infusion-related reaction (four [2%] patients) and increased lipase level (three [2%]). 16 (9%) of 184 patients had a serious adverse event related to treatment with avelumab, with infusion-related reaction (in four [2%] patients) and dyspnoea (in two [1%]) occurring in more than one patient. Serious adverse events irrespective of cause occurred in 80 (44%) of 184 patients. Those occurring in more than five patients (≥3%) were dyspnoea (ten patients [5%]), pneumonia (nine [5%]), and chronic obstructive pulmonary disease (six [3%]). Immune-related treatment-related events occurred in 22 patients (12%). Of 184 patients, 22 (12% [95% CI 8-18]) achieved a confirmed objective response, including one complete response and 21 partial responses. 70 (38%) had stable disease. Overall, 92 (50%) of 184 patients achieved disease control (they had a confirmed response or stable disease as their best overall response). One patient was initially thought to have died from grade 5 radiation pneumonitis during the study; however, this adverse event was subsequently regraded to grade 3 and the death was attributed to disease progression. INTERPRETATION Avelumab showed an acceptable safety profile and antitumour activity in patients with progressive or treatment-resistant NSCLC, providing a rationale for further studies of avelumab in this disease setting. FUNDING Merck KGaA and Pfizer.

Inhibition of the receptor tyrosine kinase Axl impedes activation of the FLT3 internal tandem duplication in human acute myeloid leukemia: implications for Axl as a potential therapeutic target

Approximately 20% to 25% of patients with acute myeloid leukemia (AML) have a constitutively activated FLT3-internal tandem duplication (FLT3-ITD), and these patients exhibit a poor prognosis. Here, we report that Axl, a receptor tyrosine kinase (RTK) overexpressed and constitutively active in human AML, targets the RTK FLT3 in FLT3-ITD(+) AML. Abrogation of Axl activation by soluble Axl chimeric protein (Axl-Fc) or small interfering RNA (siRNA) diminishes constitutive FLT3 phosphorylation in FLT3-ITD(+) AML. In addition, inhibition of Axl activation by Axl-Fc interferes with the physical interaction between Axl and FLT3. We found that Axl-Fc, a pharmacologic Axl inhibitor, or siRNA targeting Axl inhibits cell growth, induces cell-cycle arrest and apoptosis, and relieves a block in myeloid differentiation of FLT3-ITD(+) AML in vitro. Axl-Fc also suppresses the growth of human FLT3-ITD(+) AML in vivo. Collectively, our data suggest that Axl contributes to the pathogenesis of FLT3-ITD(+) AML through, at least in part, positive regulation of constitutive FLT3 activation. This also suggests that Axl should be pursued as a potential target for the treatment of FLT3-ITD(+) AML.

Phase 1/2 Study of the Safety and Tolerability of Nivolumab Plus Crizotinib for the First-Line Treatment of Anaplastic Lymphoma Kinase Translocation — Positive Advanced Non–Small Cell Lung Cancer (CheckMate 370)

Introduction: Crizotinib, an anaplastic lymphoma kinase (ALK) inhibitor, is a first‐line treatment for ALK translocation–positive advanced non–small cell lung cancer (NSCLC); however, patients eventually progress. Immunotherapies, including the programmed death‐1 inhibitor nivolumab, have resulted in durable responses and long‐term overall survival in patients with NSCLC. We hypothesized that combining targeted therapy with immunotherapy could result in more patients with responses and/or more durable responses. Herein we report data from a study assessing nivolumab plus crizotinib in patients with previously untreated advanced ALK translocation–positive NSCLC. Methods: Group E in CheckMate 370 was a single‐arm cohort designed to evaluate the safety of first‐line nivolumab (240 mg every 2 weeks) plus crizotinib (250 mg twice daily) in patients with ALK translocation–positive NSCLC. The primary endpoint of safety would be met if ≤20% of patients discontinued treatment due to treatment‐related adverse events by week 17. Objective response rate was a secondary endpoint. A planned safety review occurred in November 2016; the data cutoff was May 26, 2017. Results: Of the first 13 patients treated with nivolumab plus crizotinib, 5 (38%) developed severe hepatic toxicities leading to the discontinuation of the combination. Of these, two patients died and the presence of severe hepatic toxicities may have contributed to death. Enrollment was closed and combination treatment discontinued due to observed grade ≥3 hepatic toxicities. Five patients (38%) had a partial response. Conclusions: These findings do not support further evaluation of nivolumab 240 mg every 2 weeks plus crizotinib 250 mg twice daily.

Unusual, spontaneous aneurysm formation in a patient being treated with ibrutinib for chronic lymphocytic leukemia.

Ibrutinib has been shown to be beneficial for B-cell malignancies with overall response rates of 71% in initial clinical trials for chronic lymphocytic leukemia (CLL), irrespective of high-risk characteristics [Byrd et al. 2013; Parmar et al. 2014]. Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor approved for the treatment of selected leukemias and lymphomas, has been associated with bleeding events [Byrd et al. 2013, 2014; Wang et al. 2013]. Kamel and colleagues illustrated that BTK inhibitors, such as ibrutinib, cause an interruption in collagen-mediated platelet aggregation, and thus increase bleeding risk [Kamel et al. 2015]. Levade and colleagues demonstrated ibrutinib’s effects on adhesion of platelets to von Willebrand factor by the disruption of collagen receptors. It was also shown that removal of the drug, and the generation of new, unaffected platelets provided a decrease in the risk of bleeding [Levade et al. 2014]. While as many as 50% (or half) of patients may experience grade 1 or 2 bleeding on ibrutinib, other studies have revealed more serious bleeding events [Jones et al. 2014]. Wang and colleagues evaluated patients with mantle cell lymphoma being treated with ibrutinib. Approximately 5% of patients were shown to have grade 3 or higher bleeding events; however, the majority of these patients had sustained some sort of trauma [Wang et al. 2013]. Burger and colleagues compared ibrutinib with chlorambucil as initial therapy for patients with CLL. The median treatment duration with ibrutinib was 17.4 months, and major hemorrhage was reported in 4% of these patients [Burger et al. 2015]. Despite a noted disruption in platelet function, ibrutinib has been shown to improve overall platelet counts with the control of the underlying CLL [Farooqui et al. 2012]. Jones and colleagues evaluated the concomitant use of antiplatelet agents and anticoagulants in patients treated with ibrutinib, and the majority of the patients having major bleeding were treated with one of these agents, in addition to the BTK inhibitor [Jones et al. 2014]. Though major bleeding events have been present in patients treated with ibrutinib, the role of vascular remodeling has not previously been described. We present the case of a patient with an unusual bleeding event secondary to a vascular anomaly on ibrutinib for the treatment of CLL. The patient is a 46-year-old man treated for CLL with ibrutinib with no other medical history. He had no prior lines of therapy for his disease. At presentation he had diffuse lymphadenopathy, lymphocytosis with a white blood count of 35,000 mm3, splenomegaly and platelet count of 65,000 mm3. The patient had Rai stage IV CLL at diagnosis which was his indication for treatment. Three months after starting therapy, he experienced a dramatic positive response both in lymph node size and peripheral blood counts. WBC did increase to 55,700 mm3 within a month of treatment but had normalized to 5600 mm3 within 3 months. However, he was admitted to the hospital 3 months after initiating therapy with a severe headache and found to have a subarachnoid hemorrhage on CT and magnetic resonance imaging. Ibrutinib was discontinued upon hospitalization. He was not on any home medications including antiplatelet agents or anticoagulants at the time of this event, nor was any trauma experienced prior to admission. He had no prior history of bleeding diathesis. His prothrombin time (PT), partial thromboplastin time (PTT) and fibrinogen were within normal limits and his platelet count was 99,000 mm3, which was a significant improvement from his pretreatment platelet count of 65,000 mm3. His pretreatment hemoglobin (Hb) was 12.6 g/dl and had normalized to 15.3 g/dl within 3 months of treatment. Hb was stable throughout hospitalization averaging 15 g/dl, as well as platelet count, with no evidence of autoimmune hemolysis. Initial angiography was normal, but 5 days later repeat angiography demonstrated slow filling of an unusual aneurysm arising from the basilar artery demonstrated by the blue arrow in panels A and B of Figure 1. Three days later, a third angiogram was performed which showed spontaneous resolution of the aneurysm. Ibrutinib was held during the patient’s hospitalization and he ultimately recovered. He had no further bleeding episodes. We hypothesize that ibrutinib may interact with the collagen component of vasculature, leading to abnormal remodeling which may be associated with the increased risk of bleeding described with this agent. Figure 1. Angiogram performed on day 8 of hospitalization demonstrating unusual aneurysm. The blue arrow in panels A and B indicates location of this abnormal aneurysm from different views. This aneurysm was not present on initial angiography and was absent during ... Some researchers have used this known disruption in collagen structure and function as a beneficial side effect of ibrutinib. Dubovsky and colleagues described using the drug to deliberately interrupt collagen’s role in forming the fibrotic tissue changes that contribute to chronic graft-versus-host disease [Dubovsky et al. 2014]. The aforementioned patient likely did not have bleeding that was secondary to disrupted platelet aggregation, but rather, we hypothesize that ibrutinib may have caused some intermittent disruption or remodeling of the collagen fibers comprising his vasculature. This intermittent nature of disruption could explain his lack of initial aneurysm at presentation, spontaneous aneurysm formation, and its subsequent resolution with removal of the drug. The package insert for ibrutinib quotes a 6% grade 3 or higher bleeding risk in patients treated with ibrutinib, with this risk increased in patients on other anticoagulants or antiplatelet therapy (ibrutinib package insert, Pharmacyclics, Sunnyvale, CA, 2013). As the mechanism for bleeding events secondary to ibrutinib is not completely understood, this case illustrates that abnormal vascular remodeling leading to aberrations in normal vasculature could be a component of these hemorrhagic events in addition to disruption in platelet aggregation.

Abstract P6-11-10: IBL2001: Phase I/II study of a novel dose-dense schedule of oral indibulin for the treatment of metastastic breast cancer.

Background: Indibulin (ZI0-301) is a novel, oral, synthetic small molecule microtubule inhibitor which binds tubulin at a different site than taxanes and vinca alkaloids. Preclinical data demonstrate indibulin does not interact with acetylated (neuronal) tubulins and in clinical studies has not exhibited the neurotoxicity associated with other tubulin binders. Indibulin has potent antitumor activity in human cancer cell lines, including multidrug-, taxane-, and vinblastine-resistant. Norton-Simon modeling based on cell line data suggested that dose dense (dd) administration could optimize efficacy while limiting toxicity. Methods: Eligible patients (pts) have metastatic or unresectable locally advanced breast cancer, ECOG performance status ≤ 2, adequate organ function, measurable or nonmeasurable disease and any number of prior therapies. Uncontrolled gastrointestinal malabsorption syndrome and grade 2 or higher peripheral neuropathy are the principal exclusions. Adverse events (AEs) are graded by CTCAE v. 4.0. Objective disease status is evaluated according to RECIST 1.1. The primary objective of the phase (Ph) I portion of the study is to determine the maximum tolerated dose (MTD) of indibulin when given in dd fashion 5 days treatment, 9 days rest using standard 3+3 dose escalation schema. The secondary objectives are to evaluate safety profile at various dosing levels, pharmacokinetics (PK) and preliminary activity of indibulin. Once the MTD is defined, a food effect cross- over group (N = 12) will be enrolled. Two groups of 6 pts each will be treated in either the fed or fasted state during the first cycle. A subgroup of 13 pts consisting of 12 pts from the food effect group plus the last pt from the MTD cohort will be evaluated for PFS at 4 months and will serve as the population for the first stage of a Simon two-stage design. If 4 or more out of 13 pts do not progress at 4 months, the Ph II portion of the study will be opened. Results: Twenty one pts (20 F, 1 M) have been enrolled to cohorts 1 through 6 and the dose escalation is ongoing. Preliminary safety and efficacy data have been analyzed for 18 pts treated in cohorts 1 through 5 and are presented henceforth. No DLT has been observed and no MTD has been reached. Median age 58 years (32–81). PS 0=4, 1=12, 2=2. Median number of prior therapies 5 (1–12). Most frequent treatment-emergent AEs were: anorexia, constipation, cough, nausea (each in 39% pts); dyspnea (33%); fatigue, vomiting (each 28%). There were no related grade 3–4 AEs. PK analysis revealed that indibulin plasma exposures increased approximately dose proportionally from 25 to 200 mg with C max of 165 ± 89 ng/mL and AUC 0-24 of 1411 ± 111 ng·h/mL at 200 mg. There were no objective responses. Stable disease was seen in 1 pt in the 150 mg cohort. Longest duration on-study was 4 months. Conclusions: Oral indibulin was well tolerated in the doses up to 200 mg and the dose-proportional PK with lack of DLTs allows for further dose-escalation. Stable disease observed at sub-MTD dose may be a sign of activity in this heavily pre-treated population. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-10.

Abstract 1950: Suppression of RISC-independent decoy and RISC-mediated RNA-pairing activities of microRNA-328 is required for maturation-arrest and enhanced survival of blast crisis CML progenitors

MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind to mRNA in a sequence-specific manner. We showed that hnRNP-E2 inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BC) by suppressing CEBPA mRNA translation. We report here that loss of miR-328 is induced by BCR/ABL and specifically occurs in CML-BC, and its restored expression rescues differentiation and impairs clonogenic potential of BCR/ABL + BM progenitors. Accordingly, miR-328 increases during granulocytic differentiation of human CD34 + and mouse LSK BM stem/progenitor cells. Mechanistically, BCR/ABL uses the MAPK-hnRNP-E2 pathway to suppress C/EBPα and miR-328 expression as pharmacologic inhibition of and/or shRNAs against these molecules efficiently restore miR-328 expression. Interestingly, two functional C/EBPα binding sites are present in the miR-328 promoter and positively regulate its transcription. We also show that maturation of differentiation-arrested BCR/ABL + blasts requires direct interaction of hnRNP-E2 with the miR-328 C-rich regions. Moreover, imatinib treatment restores miR-328 expression, thus allowing its direct binding to hnRNP E2 independent from the RISC complex. Importantly, physiological miR-328 expression decreased hnRNP E2 binding to the uORF/spacer region of endogenous CEBPA mRNA (decoy activity). This, in turn, releases CEBPA mRNA from hnRNP E2 translation inhibition and allows in vitro and in vivo BCR/ABL + cell differentiation. Although hnRNP E2 was not found in complex with the basic RISC components in BCR/ABL + cells, miR-328 was found associated to Dicer and Ago2, suggesting that miR-328 also acts through base-pairing with the 3′UTR of mRNA targets in a RISC-dependent manner. In fact, miR-328 suppresses PIM1 protein but not mRNA expression and this effect requires the integrity of the PIM1 3′UTR. Indeed, forced expression of a wild type, but not a kinase-deficient, PIM1 lacking the 3′UTR into miR-328-expressing cells fully rescues BCR/ABL clonogenicity, suggesting that miR-328-induced inhibition of PIM1 accounts for reduced survival of miR-328-infected CML-BC CD34+ blasts. To demonstrate that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 in the seed sequence (miR-328-Mut) while retaining its C-rich character. As expected, miR-328-Mut interacted with hnRNP-E2 and rescued C/EBPα-mediated differentiation, but did not silence PIM1 expression. Thus, the discovery of dual activities for miR-328 which affect myeloid differentiation and survival not only adds a layer to the complexity of mechanisms regulating CML-BC but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA binding proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1950.

Correction to: Genomic landscape of small cell carcinoma of the breast contrasted to small cell carcinoma of the lung

In the original publication, the sixth author name was published incorrectly as Matthew Stein. The correct author name should read as Matthew K Stein.