The delay in naïve T cell division is organism-wide.

<p>Mice containing approximately 1.4×10⁵ CFSE-labeled SMARTA CD4⁺ T cells were infected with LCMV. At the indicated times after infection, lymphocytes were isolated (2 mice per time point) and the donor cells were identified by flow cytometry. A. The ovals in the dot plots identify the SMARTA CD4⁺ T cells, and the numbers indicate their percentage among leukocytes isolated from each tissue. The histograms show the CFSE-fluorescence of the SMARTA CD4⁺ T cells; the numbers indicate the percentages of SMARTA CD4⁺ T cells that have divided. B. The line graphs show the percentages of SMARTA CD4⁺ T cells among all isolated leukocytes at various times after infection. For each tissue, the dashed line indicates the number of SMARTA cells in uninfected mice.</p

Naive antiviral CD4⁺ and CD8⁺ T cell division has a lag phase of 2–3 days.

<p>Equal numbers of CFSE-labeled P14 cells (TcR-transgenic CD8⁺ T cells specific for LCMV GP_33–41, expressing Thy1.1) and CFSE-labeled SMARTA cells (TcR transgenic CD4⁺ T cells specific for LCMV GP_61–80, expressing Ly5a) were pooled, and inoculated into wildtype C57BL/6 mice, which then were infected with LCMV. A. At the indicated times after infection, each donor population was identified by flow cytometry (ovals). B. The numbers of P14 and SMARTA T cells in the spleen are shown (mean±SE) at the indicated times after infection (two separate experiments, two mice per experiment). C. After gating to identify the P14 or the SMARTA T cells, the histograms show these cells' CFSE fluorescence. Note that both T cell subsets begin proliferating at the same time (day 3).</p

Delayed accumulation of naive and memory CD4⁺ T cells occurs also in non-lymphoid tissues.

<p>Mice containing 1.3×10⁴ naïve (Thy1.1) and 1.3×10⁴ memory (Ly5a) SMARTA CD4⁺ T cells were infected with LCMV and, at the indicated times after infection, lymphocytes from several lymphoid and non-lymphoid tissues were isolated and analyzed by flow cytometry. A. Dot plots show gated CD4⁺ T cells isolated from the tissues. The ovals identify the SMARTA cells (N, M  =  naïve & memory respectively), and the numbers indicate their percentage among all CD4 T cells (H  =  host CD4⁺ T cells). B. For each tissue, naïve and memory SMARTA CD4⁺ T cells are shown as percentages of all CD4⁺ T cells (two mice per time point). Note that both naïve and memory cells become prominent after day 4; however, the memory cells dominate the response in the non-lymphoid tissues.</p

Kinetics of naive and memory CD4⁺ T cells in the same mouse.

<p>Wildtype mice containing 1.3×10³ naïve SMARTA (Thy1.1) and 1.3×10³ memory SMARTA (Ly5a) cells were given LCMV, and the relative abundance of the two SMARTA cell populations was determined by flow cytometry at various times post infection (two mice per time point). A. After gating on CD4⁺ T cells, the host CD4⁺ T cells (H), and the naïve and memory SMARTA cells (N & M respectively) were distinguished by Thy1.1 and Ly5a staining. The numbers indicate the frequencies of naïve and memory SMARTA cells as a percentage of all CD4⁺ T cells. B. The average±SE of the percentage of each population among all CD4⁺ T cells is shown over time. C. The total number of memory or naïve SMARTA CD4⁺ T cells per spleen is shown (average±SE).</p

Changing the microenvironment reduces the in vivo delay in T cell division.

<p>Naive SMARTA cells were CFSE-labeled and transferred either to mice that had been infected with LCMV two days previously, or to uninfected mice some of which were immediately infected with LCMV. A. 2, 3 or 4 days after cell transfer (as indicated), the spleens of the recipient mice were isolated and the donor SMARTA CD4⁺ T cells were identified by flow cytometry (ovals). Individual mice are shown, and the numbers indicate the proportion of SMARTA cells as a percentage of all spleen cells. Mouse numbers in each of the 4 groups: 1, 1, 3, 3. B. The bar graph shows cumulative data, as percentages of SMARTA CD4⁺ T cells. C. The histograms show the CFSE fluorescence of the indicated SMARTA CD4⁺ T cells. Note that the 3-day delay in proliferation is shortened to 2 days if the mice were pre-infected.</p

Viral epitopes are presented within hours of infection, and stimulate memory T cell effector functions.

<p>Mice that contained approximately 3×10³ SMARTA/Ly5a CD4⁺ T cells were infected with LCMV and, 354 days later, were re-challenged intraperitoneally with 2×10⁶ PFU LCMV-Armstong. Six hours post-infection, the mice were given 0.25 mg Brefeldin A i.v., and 6 hours later the spleens were harvested and immediately surface stained for CD4, Ly5a, or CD8, then permeabilized and stained for intracellular IFNγ. The cells were not re-stimulated <i>ex vivo</i> with peptide antigen. A. ∼5% of all CD8⁺ T cells, and ∼1% of all CD4⁺ T cells, are actively producing IFNγ in response to infection. B. Using the SMARTA cells transferred ∼1 year previously as an indicator of the responsiveness of virus-specific CD4⁺ memory T cells, ∼14% of LCMV-specific CD4⁺ memory T cells actively produce IFNγ within 12 hours of virus infection. Data shown are from an individual mouse, and are representative of independent datasets. C. A separate set of naive mice were given CFSE-labeled pooled SMARTA cells (4×10⁵ naive SMARTA/Thy1.1 cells and 2×10⁴ memory SMARTA/Ly5a T cells). 4 days later, some of the recipient mice were given LCMV. Six hours later, BFA was administered to all mice, and after a further 6 hours splenocytes were harvested. The cells were immediately stained (without peptide re-stimulation) for CD4, Thy1.1, Ly5a and IFNγ, and were analyzed by flow cytometry. Approximately 2% memory SMARTA cells had begun to synthesize IFNγ in response to LCMV infection (top row) but none of those responding memory cells showed any dilution of CFSE signal. The naïve SMARTA cells (bottom row) failed to produce IFNγ at this early time point post-infection, and no sign of cell division was seen. Data are from one of two independent experiments.</p

Naive and memory CD4⁺ T cells show near-identical delays in onset of division.

<p>Mice containing 2×10³ naive SMARTA CD4⁺ T cells (Ly5a) were infected with LCMV and allowed to become immune. A. Six months after infection, memory SMARTA CD4⁺ T cells were isolated from the spleen and analyzed by flow cytometry. The first dot plot identifies the memory SMARTA CD4⁺ T cells (oval). After gating on these cells, the histogram shows their expression of CD44, and the remaining two dot plots evaluate IFNγ and IL-2 production after brief <i>in vitro</i> stimulation with GP_61–80 peptide. B. The memory SMARTA cells (Ly5a) were mixed with naive SMARTA cells (Thy1.1), labeled with CFSE, and then transferred to naive mice. The recipient mice were given approximately 5×10⁴ memory SMARTA CD4 T cells and 5×10⁵ naive SMARTA CD4 T cells. 3 days later, the recipients were infected with LCMV. The dot plots show spleen cells isolated from recipient mice at the indicated times after infection, and the ovals identify the memory SMARTA CD4 T cells (top two rows) and the naive SMARTA CD4 T cells (bottom two rows). The histograms show the CFSE fluorescence of the SMARTA cells, and the numbers in the histograms indicate the percentage of SMARTA CD4⁺ T cells that have not divided. Data are representative of two independent experiments.</p

DataSheet_1_Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors.docx

Oncoprotein expression is controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, a component of eIF4F, catalyzes the unwinding of secondary structure in the 5’-untranslated region (5’-UTR) of mRNA to facilitate ribosome scanning and translation initiation. Zotatifin (eFT226) is a selective eIF4A inhibitor that increases the affinity between eIF4A and specific polypurine sequence motifs and has been reported to inhibit translation of driver oncogenes in models of lymphoma. Here we report the identification of zotatifin binding motifs in the 5’-UTRs of HER2 and FGFR1/2 Receptor Tyrosine Kinases (RTKs). Dysregulation of HER2 or FGFR1/2 in human cancers leads to activation of the PI3K/AKT and RAS/ERK signaling pathways, thus enhancing eIF4A activity and promoting the translation of select oncogenes that are required for tumor cell growth and survival. In solid tumor models driven by alterations in HER2 or FGFR1/2, downregulation of oncoprotein expression by zotatifin induces sustained pathway-dependent anti-tumor activity resulting in potent inhibition of cell proliferation, induction of apoptosis, and significant in vivo tumor growth inhibition or regression. Sensitivity of RTK-driven tumor models to zotatifin correlated with high basal levels of mTOR activity and elevated translational capacity highlighting the unique circuitry generated by the RTK-driven signaling pathway. This dependency identifies the potential for rational combination strategies aimed at vertical inhibition of the PI3K/AKT/eIF4F pathway. Combination of zotatifin with PI3K or AKT inhibitors was beneficial across RTK-driven cancer models by blocking RTK-driven resistance mechanisms demonstrating the clinical potential of these combination strategies.</p

Supplementary Data from Targeting Oncogene mRNA Translation in B-Cell Malignancies with eFT226, a Potent and Selective Inhibitor of eIF4A

Tables S1-S3, Figures S1-S5</p

Structure-based Design of Pyridone–Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)­1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound <b>23</b> (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone–aminal structure described for the first time in the kinase literature. Compound <b>23</b> has potent <i>in vivo</i> antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound <b>23</b> is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound <b>23</b> is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically