Noncanonical microprotein regulation of immunity.

The immune system is highly regulated but, when dysregulated, suboptimal protective or overly robust immune responses can lead to immune-mediated disorders. The genetic and molecular mechanisms of immune regulation are incompletely understood, impeding the development of more precise diagnostics and therapeutics for immune-mediated disorders. Recently, thousands of previously unrecognized noncanonical microprotein genes encoded by small open reading frames have been identified. Many of these microproteins perform critical functions, often in a cell- and context-specific manner. Several microproteins are now known to regulate immunity; however, the vast majority are uncharacterized. Therefore, illuminating what is often referred to as the "dark proteome," may present opportunities to tune immune responses more precisely. Here, we review noncanonical microprotein biology, highlight recently discovered examples regulating immunity, and discuss the potential and challenges of modulating dysregulated immune responses by targeting microproteins.

PI3K Targeting in Non-solid Cancer.

Despite the therapeutic progress, relapse remains a major problem in the treatment of acute lymphoblastic leukemia (ALL). Most leukemia cells that survive chemotherapy are found in the bone marrow (BM), thus resistance to chemotherapy and other treatments may be partially attributed to pro-survival signaling to leukemic cells mediated by leukemia cell-microenvironment interactions. Adhesion of leukemia cells to BM stromal cells may lead to cell adhesion-mediated drug resistance (CAM-DR) mediating intracellular signaling changes that support survival of leukemia cells. In ALL and chronic lymphocytic leukemia (CLL), adhesion-mediated activation of the PI3K/AKT signaling pathway has been shown to be critical in CAM-DR. PI3K targeting inhibitors have been approved for CLL and have been evaluated preclinically in ALL. However, PI3K inhibition has yet to be approved for clinical use in ALL. Here, we review the role of PI3K signaling for normal hematopoietic and leukemia cells and summarize preclinical inhibitors of PI3K in ALL.

Preclinical Evaluation of a Novel Dual Targeting PI3Kδ/BRD4 Inhibitor, SF2535, in B-Cell Acute Lymphoblastic Leukemia.

The PI3K/Akt pathway-and in particular PI3Kδ-is known for its role in drug resistant B-cell acute lymphoblastic leukemia (B-ALL) and it is often upregulated in refractory or relapsed B-ALL. Myc proteins are transcription factors responsible for transcribing pro-proliferative genes and c-Myc is often overexpressed in cancers. The chromatin regulator BRD4 is required for expression of c-Myc in hematologic malignancies including B-ALL. Previously, combination of BRD4 and PI3K inhibition with SF2523 was shown to successfully decrease Myc expression. However, the underlying mechanism and effect of dual inhibition of PI3Kδ/BRD4 in B-ALL remains unknown. To study this, we utilized SF2535, a novel small molecule dual inhibitor which can specifically target the PI3Kδ isoform and BRD4. We treated primary B-ALL cells with various concentrations of SF2535 and studied its effect on specific pharmacological on-target mechanisms such as apoptosis, cell cycle, cell proliferation, and adhesion molecules expression usingin vitro and in vivo models. SF2535 significantly downregulates both c-Myc mRNA and protein expression through inhibition of BRD4 at the c-Myc promoter site and decreases p-AKT expression through inhibition of the PI3Kδ/AKT pathway. SF2535 induced apoptosis in B-ALL by downregulation of BCL-2 and increased cleavage of caspase-3, caspase-7, and PARP. Moreover, SF2535 induced cell cycle arrest and decreased cell counts in B-ALL. Interestingly, SF2535 decreased the mean fluorescence intensity (MFI) of integrin α4, α5, α6, and ß1 while increasing MFI of CXCR4, indicating that SF2535 may work through inside-out signaling of integrins. Taken together, our data provide a rationale for the clinical evaluation of targeting PI3Kδ/BRD4 in refractory or relapsed B-ALL using SF2535.

Risk-adapted, ofatumumab-based chemoimmunotherapy and consolidation in treatment-naïve chronic lymphocytic leukemia: a phase 2 study.

High-risk cytogenetics and minimal residual disease (MRD) after chemoimmunotherapy (CIT) predict unfavorable outcome in chronic lymphocytic leukemia (CLL). This phase 2 study investigated risk-adapted CIT in treatment-naïve CLL (NCT01145209). Patients with high-risk cytogenetics received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab consolidation. MRD negative (MRD-) patients had no intervention. Of 28 evaluable for response, all responded to induction and 10 (36%) achieved MRD-. Two-year progression-free survival (PFS) was 71.4% (CI95, 56.5-90.3%). There was no significant difference in median PFS between the high-risk and the standard-risk groups. Ofatumumab consolidation didn't convert MRD + to MRD-. In the MRD + group, we saw selective loss of CD20 antigens during therapy. In conclusion, risk-adapted CIT is feasible in treatment-naïve CLL. Ofatumumab consolidation didn't improve depth of response in MRD + patients. Loss of targetable CD20 likely reduces efficacy of consolidation therapy.

Targeting PI3K Signaling in Acute Lymphoblastic Leukemia.

Adhesion of acute lymphoblastic leukemia (ALL) cells to bone marrow stroma cells triggers intracellular signals regulating cell-adhesion-mediated drug resistance (CAM-DR). Stromal cell protection of ALL cells has been shown to require active AKT. In chronic lymphocytic leukemia (CLL), adhesion-mediated activation of the PI3K/AKT pathway is reported. A novel FDA-approved PI3Kδ inhibitor, CAL-101/idelalisib, leads to downregulation of p-AKT and increased apoptosis of CLL cells. Recently, two additional PI3K inhibitors have received FDA approval. As the PI3K/AKT pathway is also implicated in adhesion-mediated survival of ALL cells, PI3K inhibitors have been evaluated preclinically in ALL. However, PI3K inhibition has yet to be approved for clinical use in ALL. Here, we review the role of PI3K in normal hematopoietic cells, and in ALL. We focus on summarizing targeting strategies of PI3K in ALL.

A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era.

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naïve patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naïve CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naïve patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb's and other T cell-recruiting bsAb's as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.

MALT1 Inhibition Is Efficacious in Both Naïve and Ibrutinib-Resistant Chronic Lymphocytic Leukemia.

The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Bruton's tyrosine kinase (BTK), is essential for B-cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2, a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease. Cancer Res; 77(24); 7038-48. ©2017 AACR.

Bisphenol A Induces Sox2 in ER+ Breast Cancer Stem-Like Cells.

Bisphenol A (BPA) is an endocrine disrupting compound used in food and beverage plastic containers and has been shown to increase breast cancer cellular proliferation. However, the concentrations of BPA used in these experiments are far higher than the physiological levels of BPA detected in the human body. We observed in vitro that exposure of MCF-7 cells to physiological concentrations of BPA failed to increase cell proliferation or to induce canonical estrogen-responsive genes (pS2 and progesterone receptor (PR)), in contrast to 17ß-estradiol (E2) treatment. However, MCF-7 cells treated with 10 nM BPA induced ALDH1 expression, a marker of human mammary stem cells. When treated with 10 nM BPA, mammospheres derived either from MCF-7 cells, a patient-derived xenograft, or the normal mouse mammary gland exhibited increased size; however, these effects were not observed in MDA-MB-231 mammospheres. Mechanistically, BPA induced SOX2 mRNA and protein in MCF-7 mammospheres, resulting from enhanced CREB phosphorylation, and subsequent binding of pCREB to a SOX2 downstream enhancer. These findings suggest that physiological levels of BPA increase estrogen receptor-positive breast cancer tumor maintenance through enhanced cancer stem-like cell activity via direct regulation of SOX2 transcription.

Methylparaben stimulates tumor initiating cells in ER+ breast cancer models.

A body of epidemiological evidence implicates exposure to endocrine disrupting chemicals (EDCs) with increased susceptibility to breast cancer. To evaluate the physiological effects of a suspected EDC in vivo, we exposed MCF-7 breast cancer cells and a patient-derived xenograft (PDX, estrogen receptor positive) to physiological levels of methylparaben (mePB), which is commonly used in personal care products as a preservative. mePB pellets (4.4 µg per day) led to increased tumor size of MCF-7 xenografts and ER+ PDX tumors. mePB has been thought to be a xenoestrogen; however, in vitro exposure of 10 nM mePB failed to increase MCF-7 cell proliferation or induction of canonical estrogen-responsive genes (pS2 and progesterone receptor), in contrast to 17ß-estradiol (E2) treatment. MCF-7 and PDX-derived mammospheres exhibited increased size and up-regulation of canonical stem cell markers ALDH1, NANOG, OCT4 and SOX2 when exposed to mePB; these effects were not observed for MDA-MB-231 (ER- ) mammospheres. As tumor-initiating cells (TICs) are also believed to be responsible for chemoresistance, mammospheres were treated with either tamoxifen or the pure anti-estrogen fulvestrant in the presence of mePB. Blocking the estrogenic response was not sufficient to block NANOG expression in mammospheres, pointing to a non-classic estrogen response or an ER-independent mechanism of mePB promotion of mammosphere activity. Overall, these results suggest that mePB increases breast cancer tumor proliferation through enhanced TIC activity, in part via regulation of NANOG, and that mePB may play a direct role in chemoresistance by modulating stem cell activity. Copyright © 2016 John Wiley & Sons, Ltd.

Serum B-cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival.

Although TNFRSF17 (also designated as B-cell maturation antigen (BCMA)) is expressed on tumour cells in B-cell malignancies, it has not been found in serum. The present study found that BCMA concentrations were higher in the supernatants of cultured bone marrow mononuclear cells from multiple myeloma (MM) patients than in healthy subjects. Serum BCMA levels were measured in samples from MM patients (n = 209), monoclonal gammopathy of undetermined significance (MGUS) individuals (n = 23) and age-matched controls (n = 40). BCMA was detected in the serum of untreated MM patients (n = 50) and levels were higher than in MGUS patients (P = 0·0157) and healthy subjects (P < 0·0001). Serum BCMA levels were higher among patients with progressive disease (n = 80) compared to those with responsive disease (n = 79; P = 0·0038). Among all MM patients, overall survival was shorter among patients whose serum BCMA levels were above the median (P = 0·001). We also demonstrated that sera from mice with human MM xenografts contained human BCMA, and levels correlated with the change in tumour volume in response to melphalan or cyclophosphamide with bortezomib. These results suggest that serum BCMA levels may be a new biomarker for monitoring disease status and overall survival of MM patients.

Anti-myeloma effects of the novel anthracycline derivative INNO-206.

PURPOSE: Doxorubicin has shown efficacy especially in combination treatment for the treatment of multiple myeloma; however, its side effects limit its use. INNO-206 is an albumin-binding prodrug of doxorubicin, which is released from albumin under acidic conditions. Because INNO-206 has not been previously evaluated in any hematologic malignancy, we determined its anti-multiple myeloma effects. EXPERIMENTAL DESIGN: The anti-multiple myeloma effect of INNO-206 at different pH levels on multiple myeloma cell proliferation using multiple myeloma cell lines with the MTS assay and antiangiogenic activity using the chorioallantoic membrane/feather bud assay were determined. The anti-multiple myeloma effects and toxicity of INNO-206 were also compared with conventional doxorubicin and PEGylated liposomal doxorubicin (PLD) alone, and in combination with bortezomib, using our multiple myeloma xenograft models. RESULTS: INNO-206 inhibited blood vessel formation and reduced multiple myeloma cell growth in a pH-dependent fashion. INNO-206 alone produced marked anti-multiple myeloma effects in vivo at doses that doxorubicin was toxic, and the combination of INNO-206 plus bortezomib produced increased anti-multiple myeloma effects compared with either agent alone. In contrast, all mice receiving bortezomib with doxorubicin or PLD died. CONCLUSIONS: These findings show that INNO-206 produces anti-multiple myeloma effects in vitro and in vivo. It also enhances the antitumor effects of bortezomib. These results suggest that INNO-206 may provide patients with multiple myeloma with an anthracycline that may be administered safely at higher doses compared with free doxorubicin, resulting in superior efficacy compared with the currently available anthracyclines to treat this B-cell malignancy.