Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies.

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.

Mutations in the thymidine kinase gene that allow expression of the enzyme in quiescent (G0) cells.

Thymidine kinase (TK) is a nucleotide salvage pathway enzyme whose activity is highly dependent on the growth state and cell cycle phase of a cell. Cells in the resting or quiescent (G0) phase express very low levels of TK mRNA and protein. When quiescent cells are stimulated to enter the cell cycle by the addition of serum, TK mRNA, activity and polypeptide increase coordinately after about 10-15 h, at the beginning of S phase. When growth-independent heterologous promoters are substituted for the natural TK promoter, TK mRNA can be expressed in quiescent cells. Despite the presence of TK mRNA in such G0 cells, there is little expression of TK polypeptide; the normal increase in enzyme at S phase is observed following serum stimulation. Deletion of the introns and 3' untranslated sequences does not affect the expression of the TK gene in serum stimulation experiments. In contrast, deletion of the C-terminal 40 amino acids or fusion of a small segment of a beta-galactosidase to the C-terminus overcomes the block to expression of the TK polypeptide in G0 cells. These C-terminal alterations are the same as those which lead to constitutive expression of TK during the cell cycle of proliferating cells, suggesting that mechanisms which control the levels of TK in cycling cells may also operate in quiescent cells.

Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis.

The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme.

Isolation of cell cycle fractions by counterflow centrifugal elutriation.

Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 X 10(8) cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing greater than 95% G1, greater than 80% S, and greater than 70% G2/M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.

Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A.

We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.

Accessory cells in immune suppression. II. Evidence for presentation of idiotype-specific suppressor factors to B cell targets by I-A+ accessory cells.

The functional suppression of two BALB/c myelomas, MOPC315 and MOPC460, by idiotype-specific suppressor T cells is mediated by secreted factors (TsF). Idiotype-specific TsF only functions in the presence of accessory cells (AC) that can be low density splenocytes or cells of BALB/c B lymphoma A20-2J. Such AC are I-A+ but do not have to be histocompatible with the myeloma targets or the source of TsF. A20-2J cells incubated with soluble idiotype and TsF also are capable of suppressing the relevant myeloma target. The effect of TsF-pulsed A20-2J cells is idiotype specific, and these cells do not secrete immunosuppressive mediators. Finally, TsF-pulsed A20-2J cells suppress myeloma targets when the two are cultured on the same side of a cell-impermeable membrane but not when they are separated by the membrane. These results indicate that I-A+ cells are capable of concentrating and/or presenting TsF to suppressible targets, and they demonstrate a novel role for Ia-bearing cells in immune regulation.