Autophagy refers to the catabolic process in eukaryotic cells that delivers cytoplasmic material to lysosomes for degradation. This highly conserved process is involved in the clearance of long-lived proteins and damaged organelles. Consequently, autophagy is important in providing nutrients to maintain cellular function under starvation, maintaining cellular homeostasis, and promoting cell survival under certain conditions. Several pathways, including mTOR, have been shown to regulate autophagy. However, the impact of lysosomal function impairment on the autophagy process has not been fully explored. Basic lipophilic compounds can accumulate in lysosomes via pH partitioning leading to perturbation of lysosomal function. Our hypothesis is that these types of compounds can disturb the autophagy process. Eleven drugs previously shown to accumulate in lysosomes were selected and evaluated for their effects on cytotoxicity and autophagy using ATP depletion and LC3 assessment, respectively. All eleven drugs induced increased staining of endogenous LC3 and exogenous GFP-LC3, even at non toxic dose levels. In addition, an increase in the abundance of SQSTM1/p62 by all tested compounds denotes that the increase in LC3 is due to autophagy perturbation rather than enhancement. Furthermore, the gene expression profile resulting from in vitro treatment with these drugs revealed the suppression of plentiful long-lived proteins, including structural cytoskeletal and associated proteins, and extracellular matrix proteins. This finding indicates a retardation of protein turnover which further supports the notion of autophagy inhibition. Interestingly, upregulation of genes containing antioxidant response elements, e.g. glutathione S transferase and NAD(P)H dehydrogenase quinone 1 was observed, suggesting activation of Nrf2 transcription factor. These gene expression changes could be related to an increase in SQSTM1/p62 resulting from autophagy deficiency. In summary, our data indicate that lysosomal accumulation due to the basic lipophilic nature of xenobiotics could be a general mechanism contributing to the perturbation of the autophagy process.

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BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0435-x) contains supplementary material, which is available to authorized users.

Key Points PF-06747143, a novel CXCR4 antagonist IgG1 Ab, mobilizes malignant cells from the BM and induces their death via Fc-effector function. PF-06747143 reduces tumor burden in NHL, AML, and MM models, both as a monotherapy or in combination with standard-of-care agents.

The ocular cell surface inflammatory biomarker, HLA-DR coupled with impression cytology is a simple non-invasive robust, specific and reproducible assay that can be utilized to measure inflammatory infiltrates on the surface of the eye in IC samples less than 10-days old.

Kidney injury biomarkers have been utilized by pharmaceutical companies as a means to assess the potential of candidate drugs to induce nephrotoxicity. Multiple platforms and assay methods exist, but the comparison of these methods has not been described. Millipore's Kidney Toxicity panel, EMD/Novagen's Widescreen Kidney Toxicity panel, and Meso Scales Kidney Injury panel were selected based on published information. Kidney injury molecule 1, cystatin C, clusterin, and osteopontin were the 4 biomarkers common among all kits tested and the focus of this study. Rats were treated with a low and high dose of para-aminophenol, a known nephrotoxicant, and urine samples were collected and analyzed on the Bio-Plex 200 or MSD's Sector Imager 6000, according to manufacturers specifications. Comparatively, of the 3 kits, Millipore was the most consistent in detecting elevations of 3 out of the 4 biomarkers at both dose levels and indicated time points.

Background: INO-VATE (NCT01564784) demonstrated higher rates of remission, MRD-negativity, and improved overall survival (hazard ratio 0.75, one-sided P=0.01) for pts with R/R ALL who received InO vs standard chemotherapy (SC). Here we explore the potential association between leukemic molecular profiles and efficacy of InO. Methods: Pts with CD22+ ALL due to receive salvage treatment were randomized to InO or SC; clinical methods and results were published previously (Kantarjian HM, et al. N Engl J Med 2016; PMID: 27292104). Bone marrow samples collected at screening post protocol amendment 2 underwent RNA-sequencing with analysis of gene expression profile, fusions, and genomic alterations. Data cutoff: Jan 4, 2017. Results: Of 326 pts in the INO-VATE intent-to-treat (ITT) population, 91 (InO, 43; SC, 48) had samples evaluable for molecular profiling. Baseline characteristics were similar in the evaluable pts vs the ITT population. Most common (≥5%) ALL subtypes were BCR-ABL1-positive (14%), low hypodiploidy (12%), Philadelphia chromosome (Ph)-like (11%), KMT2A-rearranged (9%), and DUX4-rearranged (6%); 29% (26/91) of pts could not be subtyped due to low blast counts. Most common (≥5%) non-BCR-ABL1 oncogenic fusions detected were IGH-CRLF2 (8%), and KMT2A-AFF1 (6%). Among non-fusion genomic alterations, those involving chromatin modifying genes featured prominently (e.g. KMT2D, 18%; CREBBP, 12%; KMT2C, 11%; ARID2, 9%; ARID1A, 8%; SET2D, 7%; EP300, 6%). Other commonly altered (≥9%) genes included TP53 (14%), NOTCH1 (11%), KRAS (9%), and PAX5 (9%). Similar to the overall findings of INO-VATE, complete remission (CR)/CR with incomplete count recovery (CRi) rates were numerically higher with InO vs SC across ALL subtypes, including but not limited to, low hypodiploidy (67% [2/3] vs 13% [1/8], P=0.15), Ph-like (83% [5/6] vs 0% [0/4], P=0.02), and KMT2A (100% [2/2] vs 17% [1/6], P=0.11), although not in BCR-ABL1-positive (67% [4/6] vs 57% [4/7], P=0.59). Within pts bearing common non-BCR-ABL1 oncogenic fusions, CR/CRi was seen for InO including for IGH-CRLF2 (50% [1/2] vs 20% [1/5] for SC), KMT2A-AFF1 (100% [1/1] vs 25% [1/4] for SC), and IGH-DUX4 (100% [3/3] vs 0% [0/1] for SC). Consistent with a previous report (Yang X, et al. Blood 2019; 134 [Suppl 1]: 3888) CR/CRi rates were high for InO in pts with TP53 alterations (100% [5/5] vs 13% [1/8] for SC, P=0.0047). Conclusions: This retrospective, exploratory analysis of INO-VATE demonstrated potential for benefit with InO for ALL pts across leukemic subtypes, including Ph-like, and for those bearing diverse fusions. Further exploration of Ph-like subtype or TP53 alterations as candidate predictive biomarkers for InO in pts with R/R ALL is warranted. Citation Format: Yaqi Zhao, A Douglas Laird, Kathryn Roberts, Rolla Yafawi, Hagop Kantarjian, Daniel J. DeAngelo, Matthias Stelljes, Michaela Liedtke, Wendy Stock, Nicola Gökbuget, Susan O'Brien, Elias Jabbour, Ryan D. Cassaday, Melanie R. Loyd, Scott Olsen, Geoffrey Neale, Xueli Liu, Erik Vandendries, Charles G. Mullighan, Anjali Advani. Exploration of association of leukemic molecular profile with efficacy in patients (pts) with relapsed/refractory acute lymphoblastic leukemia (ALL) treated with inotuzumab ozogamicin (InO) in the phase 3 INO-VATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT027.

The principle of Impression Cytology (IC) utilizes a non-invasive technique to remove superficial layers of the ocular surface. The application of a filter to the surface of the bulbar conjunctiva allows for repeated collection of superficial cells for the analysis of ocular surface disorders. Evidence suggests expression of the HLA-DR antigen on the surface of conjunctival epithelial cells is associated with allergic conjunctivitis and dry eye syndrome. Performance characteristics of HLA-DR was evaluated through intra, inter and stability assays. Evaluation of the reproducibility and overall performance of the novel methodology would be useful in understanding the tolerance level of the assay. Utilizing established techniques, IC specimens were obtained from 12 donors and assayed over multiple days and timepoints. Conjunctival epithelial cells were extracted and assayed based on differential immunostaining of both conjunctival epithelial cells and leukocytes. The reproducibility across donors over time was consistent, demonstrating conjunctival cells maintained their integrity of cell surface expression of HLA-DR. In conclusion, the studies met all validation criteria, and support the utilization of HLA-DR inflammatory measurements in impression cytology specimens. Utilization of a flow cytometric platform to examine the expression of HLA-DR antigen may help predict clinical outcome, evaluate disease progression, and monitor the effects of treatment.