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Background: DNA-dependent protein kinase (DNA-PK) is a critical sensor of DNA double-strand breaks (DSBs) and orchestrates their repair through non-homologous end joining (NHEJ). AZD7648 is a novel, potent and highly selective inhibitor of DNA-PK being evaluated as monotherapy or in combination with doxorubicin or olaparib in a Phase I/II first-in-human study. The objectives of this trial are to assess safety and tolerability, establish a Recommended Phase 2 Dose (RP2D) and evaluate early clinical activity. Pre-clinically, AZD7648 monotherapy leads to tumour growth inhibition in ATM deficient models in vivo, demonstrating potential to enhance the effects of endogenous tumour damage clinically. AZD7648 is also an efficient sensitizer of doxorubicin-induced DNA damage and demonstrates enhanced efficacy in combination with olaparib. Both combinations induce sustained regressions in vivo across a range of tumour xenografts with differing genetic alterations, accompanied by robust pharmacodynamic biomarker modulation. These data provide the rationale to explore AZD7648 as monotherapy and in combination with either doxorubicin or olaparib. Methods: This is a Phase I/IIa study comprising 3 modules: Core Module (AZD7648 monotherapy - dose escalation), combination module 1 (AZD7648 + pegylated liposomal doxorubicin [PLD]), and combination module 2 (AZD7648 + olaparib). Each combination evaluates AZD7648 in a Phase I escalation (Part A) and a Phase IIa expansion (Part B). Patients are assigned to a single study Part, but intra-patient dose escalation is permitted in Part A. The study incorporates a Bayesian adaptive dose escalation trial design which combines prior expectations about the dose toxicity relationship and applies the data at the end of each cohort to recommend a dose for the next cohort. The study will investigate pharmacodynamic (PD) and exploratory biomarker (DNA, RNA, proteins or metabolites) profiles in exclusive cohorts, and study their relationship to drug effect. Eligible patients are aged ≥18 years with advanced solid tumours, who have failed standard therapies. In Part A, patients are assessed for dose-limiting toxicities during the evaluable period. Blood samples are collected for pharmacokinetic (PK) analysis following a single dose of AZD7648 and at steady state. Single patient cohorts may occur for the first four cohorts (or until drug-related ≥ Grade 2 toxicity). From cohort 5 onwards, a minimum of 3 patients will be enrolled into each dose cohort until the Maximum Tolerated Dose, Maximum Feasible Dose or RP2D is identified. Enrolment into the study commenced in Oct 2019 and is currently enrolling cohort 3 in the monotherapy dose escalation. To date, no DLTs have been reported in prior cohorts. [clinicaltrials.gov identifier: NCT03907969] Citation Format: Timothy A. Yap, Yingxue Chen, Lisa H. Butler, Si-houy Lao-Sirieix, Elaine Cadogan, Lenka Oplustil O'Connor, Karthick Vishwanathan, Younghwa Kim, Dónal Landers, Emma Dean. AZD7648: A Phase I/IIa first-in-human trial of a novel, potent and selective DNA-PK inhibitor in patients with advanced malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT248.

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34+ hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34+ cells to bone marrow stroma and by negatively regulating CD34+hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34+ cells are CD164+, with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34+ human bone marrow and cord blood cells that were CD38lo/− or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34+CD71lo/− or CD34+CD33lo/− cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34+CD164(103B2/9E10)lo/−cells in bone marrow are mainly CD19+ B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34lo/−CD19+ and CD34lo/−CD20+ B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34lo/−CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21.© 1998 by The American Society of Hematology.

Chromosomal rearrangement of the HRX (MLL, ALL-1, Htrx) gene situated at chromosome band 11q23 is one of the most frequent genetic changes in infant leukemias of myeloid and lymphoid lineage and in treatment-induced secondary leukemias. The HRX gene codes for a predicted 431-kD protein that shows significant homology to the Drosophila trithorax protein, an Hox epigenetic regulator. Typically, the region encoding the HRX gene is rearranged, mostly in reciprocal translocations with a number of partners, resulting in a range of fusion genes. However, this is not the only abnormality affecting HRX because partial duplication of the gene, as well as interstitial deletions, can occur. Despite extensive studies of HRX at the genetic level, the protein products of the HRX gene and their patterns of expression in normal and leukemic cells remain uncharacterized. In this study we analyzed the distribution and localization of HRX proteins in cell lines and human tissues, using both polyclonal and monoclonal antibodies. The specificity of these reagents was confirmed using cells transfected with the HRX-ENL fusion gene. Western blot analyses of protein extracts from cells carrying the t(11; 19) and t(4; 11) translocations showed HRX chimeric proteins whose migrations corresponded to the sizes predicted from analyses of translocation-induced fusion mRNAs expressed by the derivative 11 chromosomes. Immunocytochemical analysis showed a punctate distribution of wild-type and chimeric HRX proteins within cell nuclei, suggesting that HRX localizes to nuclear structures in cells with and without 11q23 translocations. Nuclear staining was found in the majority of tissues studied with the strongest reactivity in cerebral cortex, kidney, thyroid, and lymphoid tissues. Thus, HRX is widely expressed in most cell types including hematopoietic cells, a finding that precludes an immunocytochemical approach for diagnosis of leukemias bearing 11q23 structural abnormalities.

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1–6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.