James L. LaBelle

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

Distinct BimBH3 (BimSAHB) stapled peptides for structural and cellular studies.

Hydrocarbon stapling is a chemical approach to restoring and fortifying the natural α-helical structure of peptides that otherwise unfold when taken out of context from the host protein. By iterating the peptide sequence, staple type, and sites of insertion, discrete compositions can be generated to suit a diversity of biochemical, structural, proteomic, cellular, and drug development applications. Here, we reinforce key design considerations to avoid pitfalls and maximize progress when applying stapled peptides in chemistry and biology research.

Dissection of the BCL-2 family signaling network with stabilized alpha-helices of BCL-2 domains.

The BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death and, thus, has become the focus of intensive basic science inquiry and a fundamental target for therapeutic development in oncology and other diseases. Classified based on the presence of conserved alpha-helical motifs and pro- and anti-apoptotic functionalities, BCL-2 proteins participate in a complex interaction network that determines cellular fate. The identification of BCL-2 homology domain 3 (BH3) as a critical death helix that engages and regulates BCL-2 family proteins has inspired the development of molecular tools to decode and drug the interaction network. Stabilized Alpha-Helices of BCL-2 domains (SAHBs) are structurally reinforced, protease-resistant, and cell-permeable compounds that retain the specificity of native BH3 death ligands and, therefore, serve as ideal reagents to dissect BCL-2 family interactions in vitro and in vivo. Here, we describe the in vitro and cell-based methods that exploit SAHB compounds to determine the functional consequences of BH3 interactions in regulating apoptosis.

Self-assembling peptide-based building blocks in medical applications ☆

Abstract Peptides and peptide‐conjugates, comprising natural and synthetic building blocks, are an increasingly popular class of biomaterials. Self‐assembled nanostructures based on peptides and peptide‐conjugates offer advantages such as precise selectivity and multifunctionality that can address challenges and limitations in the clinic. In this review article, we discuss recent developments in the design and self‐assembly of various nanomaterials based on peptides and peptide‐conjugates for medical applications, and categorize them into two themes based on the driving forces of molecular self‐assembly. First, we present the self‐assembled nanostructures driven by the supramolecular interactions between the peptides, with or without the presence of conjugates. The studies where nanoassembly is driven by the interactions between the conjugates of peptide‐conjugates are then presented. Particular emphasis is given to in vivo studies focusing on therapeutics, diagnostics, immune modulation and regenerative medicine. Finally, challenges and future perspectives are presented. Graphical abstract Figure. No Caption available.

Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia

Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3′-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

Characterization of a Murine NKT Cell Tumor Previously Described as an Acute Myelogenous Leukemia

C1498 is an atypical myeloid leukemia that originated in a C57BL/6 mouse and has been used as a model for acute myelogenous leukemia. In studies of the immune response to C1498, we found that this tumor contained mRNA encoding the canonical NKT cell receptor V β 8.2-V α 14J α 281. Although cell-surface phenotypic analysis showed C1498 to be negative for NK1.1, it expressed several other molecules associated with NKT cell populations, such as DX5, CD1d, CD69, CD44, CD45RB and B220. RT-PCR demonstrated that C1498 contained CD3 ϵ mRNA transcripts, but message was not found for CD4, CD8 α, or CD8 β. This indicates that C1498 falls within the double negative (CD4 − CD8 − ) NKT cell lineage. RNase protection analysis showed that C1498 expressed mRNA for IL-2, IL-15, and macrophage migration inhibitory factor (MIF). These findings suggest that C1498 should be re-classified as a NKT cell leukemia with atypical myeloid features. It may, therefore, be a novel cell line in which to study NKT cell development and serve as a model for human NKT cell malignancies.

Mantle cell lymphoma in cyclin D1 transgenic mice with Bim-deficient B cells

Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma resistant to conventional chemotherapy. Although defined by the characteristic t(11;14) translocation, MCL has not been recapitulated in transgenic mouse models of cyclin D1 overexpression alone. Indeed, several genetic aberrations have been identified in MCL that may contribute to its pathogenesis and chemoresistance. Of particular interest is the frequent biallelic deletion of the proapoptotic BCL-2 family protein BIM. BIM exerts its pro-death function via its α-helical BH3 death domain that has the dual capacity to inhibit antiapoptotic proteins such as BCL-2 and MCL-1 and directly trigger proapoptotic proteins such as the mitochondrial executioner protein BAX. To evaluate a functional role for Bim deletion in the pathogenesis of MCL, we generated cyclin D1-transgenic mice harboring Bim-deficient B cells. In response to immunization, Eμ(CycD1)CD19(CRE)Bim(fl/fl) mice manifested selective expansion of their splenic mantle zone compartment. Three distinct immune stimulation regimens induced lymphomas with histopathologic and molecular features of human MCL in a subset of mice. Thus, deletion of Bim in B cells, in the context of cyclin D1 overexpression, disrupts a critical control point in lymphoid maturation and predisposes to the development of MCL. This genetic proof of concept for MCL pathogenesis suggests an opportunity to reactivate the death pathway by pharmacologic mimicry of proapoptotic BIM.

A Familial Case of Pleuropulmonary Blastoma

PURPOSE Pleuropulmonary blastoma (PPB) is a rare intrathoracic neoplasm of early childhood arising in the lung or visceral pleura. Approximately 150 cases have been reported in the literature, with only one previously documented case of PPB in siblings. PATIENTS AND METHODS We present the case of two brothers diagnosed with PPB. RESULTS A two month-old boy with an abnormal breathing pattern was referred for evaluation of a cystic mass discovered on chest radiograph. Computed tomography (CT) of the chest was performed at our institution which revealed findings compatible with congenital cystic adenomatoid malformation (CCAM) of the right middle and lower lobes. The patient underwent urgent thoracic exploration one week later after developing severe respiratory distress. Histological examination revealed PPB type I (cystic). The patients 15-month-old brother was presumed to have a CCAM noted radiographically months earlier during an asthma exacerbation. He underwent elective cyst resection and was also found to have type I PPB. The index patient was treated with adjuvant chemotherapy due to the large size of the PPB and intraoperative spillage of cystic fluid during the emergent surgery. In contrast, the brother is being followed without adjuvant chemotherapy, given the much smaller size of the PPB, wide margins of resection, and lack of spillage. Family history included an uncle diagnosed at age 11 with an unusual form of T cell acute lymphoblastic leukemia. CONCLUSION Although PPB is known to have a familial association with other neoplasms, this case represents only the second report of PPB occurring in siblings. The importance of thoroughly investigating and resecting pulmonary cystic masses in the pediatric population is highlighted by these cases.

RNA Polymerase Tags To Monitor Multidimensional Protein–Protein Interactions Reveal Pharmacological Engagement of Bcl-2 Proteins

We report the development of a new technology for monitoring multidimensional protein-protein interactions (PPIs) inside live mammalian cells using split RNA polymerase (RNAP) tags. In this new system, a protein-of-interest is tagged with an N-terminal split RNAP (RNAPN), and multiple potential binding partners are each fused to orthogonal C-terminal RNAPs (RNAPC). Assembly of RNAPN with each RNAPC is highly dependent on interactions between the tagged proteins. Each PPI-mediated RNAPN-RNAPC assembly transcribes from a separate promoter on a supplied DNA substrate, thereby generating a unique RNA output signal for each PPI. We develop and validate this new approach in the context of the Bcl-2 family of proteins. These key regulators of apoptosis are important cancer mediators, but are challenging to therapeutically target due to imperfect selectivity that leads to either off-target toxicity or tumor resistance. We demonstrate binary (1 × 1) and ternary (1 × 2) Bcl-2 PPI analyses by imaging fluorescent protein translation from mRNA outputs. Next, we perform a 1 × 4 PPI network analysis by direct measurement of four unique RNA signals via RT-qPCR. Finally, we use these new tools to monitor pharmacological engagement of Bcl-2 protein inhibitors, and uncover inhibitor-dependent competitive PPIs. The split RNAP tags improve upon other protein fragment complementation (PFC) approaches by offering both multidimensionality and sensitive detection using nucleic acid amplification and analysis techniques. Furthermore, this technology opens new opportunities for synthetic biology applications due to the versatility of RNA outputs for cellular engineering applications.

Killing Two Cells with One Stone: Pharmacologic BCL-2 Family Targeting for Cancer Cell Death and Immune Modulation

A crucial component of regulating organismal homeostasis is maintaining proper cell number and eliminating damaged or potentially malignant cells. Apoptosis, or programed cell death, is the mechanism responsible for this equilibrium. The intrinsic apoptotic pathway is also especially important in the development and maintenance of the immune system. Apoptosis is essential for proper positive and negative selection during B- and T-cell development and for efficient contraction of expanded lymphocytes following an immune response. Tight regulation of the apoptotic pathway is critical, as excessive cell death can lead to immunodeficiency while apoptotic resistance can lead to aberrant lymphoproliferation and autoimmune disease. Dysregulation of cell death is implicated in a wide range of hematological malignancies, and targeting various components of the apoptotic machinery in these cases is an attractive chemotherapeutic strategy. A wide array of compounds has been developed with the purpose of reactivating the intrinsic apoptotic pathway. These compounds, termed BH3 mimetics are garnering considerable attention as they gain greater clinical oncologic significance. As their use expands, it will be imperative to understand the effects these compounds have on immune homeostasis. Uncovering their potential immunomodulatory activity may allow for administration of BH3 mimetics for direct tumor cell killing as well as novel therapies for a wide range of immune-based directives. This review will summarize the major proteins involved in the intrinsic apoptotic pathway and define their roles in normal immune development and disease. Clinical and preclinical BH3 mimetics are described within the context of what is currently known about their ability to affect immune function. Prospects for future antitumor immune amplification and immune modulation are then proposed.