Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

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The expression and activity of the Myc oncoprotein is frequently dysregulated in human cancers due to Myc amplification, translocation, protein stabilization and/or convergence of oncogenic signaling pathways on Myc. Therapeutic inhibition of Myc is highly desirable however, prior efforts to identify specific and direct pharmacological inhibitors of Myc function have generally failed to yield Myc-selective drug-like small molecules. Herein, we have comprehensively profiled previously reported small molecule inhibitors of the Myc-Max heterodimeric complex (10058-F4, 10074-G5, MYCMI-6, KI-MS2-001/-008 (KI-MS2), Mycro3 & JKY-2-169), using an in vitro biological screening cascade in an effort to identify and characterize bona fide tool compounds for our Myc drug discovery program. Initial cellular screening of compounds using a reporter construct containing Myc binding elements upstream of the luciferase gene and cellular viability assays using the P493-6 (Myc On / Off) system identified JKY-2-169 (JKY) and KI-MS2 as compounds with on-target activity and acceptable specificity profiles to warrant further characterization. Consistent with its reported mode of action, we confirmed JKY perturbs binding of the c-Myc/MAX heterodimer to its canonical E-box DNA sequence without causing protein-protein dissociation through biochemical and in vitro Myc:MAX interaction assays. In a cellular thermal shift assay (CETSA) using P493-6 B-cells, JKY induced thermal stabilization of c-Myc and not MAX, suggesting direct and specific cellular engagement of JKY to Myc. Consistent with this, JKY phenocopied the genetic de-induction of c-Myc in P493-6 cells, inducing G0/G1 cell-cycle arrest in a dose-dependent manner. Moreover, 3’RNA-Sequencing (3’RNA-Seq.) of MV4-11 AML cells and P493-6 cells treated with JKY demonstrated an altered transcriptional program consistent with Myc deregulation that was independent of transcriptional modulation of Myc expression. Further transcriptional profiling revealed JKY exhibits a profile with high correlation to that of the small molecule MAX inhibitor, KI-MS2; consistent with the obligate dependence of Myc on heterodimerization with MAX for transcriptional activation. Together, these results highlight JKY and KI-MS2 as suitable chemical tools for deciphering Myc biology. MecRx’s small molecule Myc inhibitor MRX-1669 was developed using several orthogonal biochemical/biophysical assays including those described above. Similar to JKY, MRX-1669 exhibits cellular Myc engagement (CETSA), and evokes a similar transcriptional profile to that of JKY and KI-MS2 (3’RNA-Seq.). MRX-1669 is a drug-like molecule that provided a benchmark for further development and medicinal chemistry optimization. Citation Format: SANG-KYU KIM, RICHARD C FOITZIK, SONGHUI LI, ANDREW VINSON, ALEX SHMAYLOV, KAREN JARVIS, SUSAN JAMES, SCOTT R WALKER, MARK YORK, BENJAMIN CAO, SUSAN K NILSSON, RICKY W JOHNSTONE. In vitro on-target and selectivity profiling of small-molecule inhibitors of the Myc/Max heterodimeric complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C02. doi:10.1158/1535-7163.TARG-19-LB-C02