Reem Malek

Reduced ribosomal protein gene dosage and p53 activation in low-risk myelodysplastic syndrome

Reduced gene dosage of ribosomal protein subunits has been implicated in 5q- myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q- syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.

p53 in the CNS Perspectives on Development, Stem Cells, and Cancer

The p53 tumor suppressor potently limits the growth of immature and mature neurons under conditions of cellular stress. Although loss of p53 function contributes to the pathogenesis of central nervous system (CNS) tumors, excessive p53 function is implicated in neural tube defects, embryonic lethality, and neuronal degeneration. Thus, p53 function must be tightly controlled. The anti-proliferative properties of p53 are mediated, in part, through the induction of apoptosis, cell cycle arrest, and senescence. Although there is still much to be learned about the role of p53 in these processes, recent evidence supports exciting new roles for p53 in a wide range of processes, including neural precursor cell self-renewal, differentiation, and cell fate decisions. Understanding the full repertoire of p53 function in CNS development and tumorigenesis may provide us with novel points of therapeutic intervention for human diseases of the CNS.

The p53 Inhibitor MDM2 Facilitates Sonic Hedgehog-Mediated Tumorigenesis and Influences Cerebellar Foliation

Disruption of cerebellar granular neuronal precursor (GNP) maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh) pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2puro), which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1+/− mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis.

Structure-Function Studies of the bHLH Phosphorylation Domain of TWIST1 in Prostate Cancer Cells

The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice.

TWIST1-WDR5-Hottip regulates Hoxa9 chromatin to facilitate prostate cancer metastasis.

TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR.

Bioluminescence Tomography–Guided Radiation Therapy for Preclinical Research

PURPOSE In preclinical radiation research, it is challenging to localize soft tissue targets based on cone beam computed tomography (CBCT) guidance. As a more effective method to localize soft tissue targets, we developed an online bioluminescence tomography (BLT) system for small-animal radiation research platform (SARRP). We demonstrated BLT-guided radiation therapy and validated targeting accuracy based on a newly developed reconstruction algorithm. METHODS AND MATERIALS The BLT system was designed to dock with the SARRP for image acquisition and to be detached before radiation delivery. A 3-mirror system was devised to reflect the bioluminescence emitted from the subject to a stationary charge-coupled device (CCD) camera. Multispectral BLT and the incomplete variables truncated conjugate gradient method with a permissible region shrinking strategy were used as the optimization scheme to reconstruct bioluminescent source distributions. To validate BLT targeting accuracy, a small cylindrical light source with high CBCT contrast was placed in a phantom and also in the abdomen of a mouse carcass. The center of mass (CoM) of the source was recovered from BLT and used to guide radiation delivery. The accuracy of the BLT-guided targeting was validated with films and compared with the CBCT-guided delivery. In vivo experiments were conducted to demonstrate BLT localization capability for various source geometries. RESULTS Online BLT was able to recover the CoM of the embedded light source with an average accuracy of 1 mm compared to that with CBCT localization. Differences between BLT- and CBCT-guided irradiation shown on the films were consistent with the source localization revealed in the BLT and CBCT images. In vivo results demonstrated that our BLT system could potentially be applied for multiple targets and tumors. CONCLUSIONS The online BLT/CBCT/SARRP system provides an effective solution for soft tissue targeting, particularly for small, nonpalpable, or orthotopic tumor models.

Therapeutic Targeting of Epithelial Plasticity Programs: Focus on the Epithelial-Mesenchymal Transition

Mounting data points to epithelial plasticity programs such as the epithelial-mesenchymal transition (EMT) as clinically relevant therapeutic targets for the treatment of malignant tumors. In addition to the widely realized role of EMT in increasing cancer cell invasiveness during cancer metastasis, the EMT has also been implicated in allowing cancer cells to avoid tumor suppressor pathways during early tumorigenesis. In addition, data linking EMT to innate and acquired treatment resistance further points towards the desire to develop pharmacological therapies to target epithelial plasticity in cancer. In this review we organized our discussion on pathways and agents that can be used to target the EMT in cancer into 3 groups: (1) extracellular inducers of EMT, (2) the transcription factors that orchestrate the EMT transcriptome, and (3) the downstream effectors of EMT. We highlight only briefly specific canonical pathways known to be involved in EMT, such as the signal transduction pathways TGFβ, EFGR, and Axl-Gas6. We emphasize in more detail pathways that we believe are emerging novel pathways and therapeutic targets such as epigenetic therapies, glycosylation pathways, and immunotherapy. The heterogeneity of tumors and the dynamic nature of epithelial plasticity in cancer cells make it likely that targeting only 1 EMT-related process will be unsuccessful or only transiently successful. We suggest that with greater understanding of epithelial plasticity regulation, such as with the EMT, a more systematic targeting of multiple EMT regulatory networks will be the best path forward to improve cancer outcomes.

RK-33 Radiosensitizes Prostate Cancer Cells by Blocking the RNA Helicase DDX3

Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G1 phase cell-cycle arrest. Conversely, the low DDX3-expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer. Cancer Res; 76(21); 6340-50. ©2016 AACR.

Mutations in Lyar and p53 are synergistically lethal in female mice.

BACKGROUND Ly-1 antibody reactive clone (LYAR) is a nucleolar zinc finger protein that has been implicated in cell growth, self-renewal of embryonic stem cells, and medulloblastoma. To test whether LYAR is critical for cell growth and development, we generated Lyar mutant mice. METHODS Mice carrying the mutant Lyar(gt) allele were generated from embryonic stem cells that contained a gene-trap insertion in the Lyar gene. Phenotypic analyses were performed on Lyar mutant mice and mouse embryonic fibroblasts. Lyar(gt/gt) mice were crossed to mice lacking the p53 tumor suppressor protein and Lyar/p53 compound mutants scored for external abnormalities. RESULTS Lyar(gt/gt) homozygotes are viable, fertile, and indistinguishable from wild type littermates. However, the growth of Lyar(+/gt) and Lyar(gt/gt) mouse embryonic fibroblasts (MEFs) was impaired, coincident with an increase in the steady-state level of p53 and a key p53 effector of growth arrest, p21, suggesting that a cellular stress response is triggered in the absence of a wild type level of LYAR. Remarkably, the majority of Lyar(+/gt) and Lyar(gt/gt) female mice lacking p53 mice failed to survive. The neural tube defect (NTD) exencephaly was observed in ≈26% and ≈61% of female Lyar(+/gt;) p53(-/-) and Lyar(gt/gt;) p53(-/-) embryos, respectively. CONCLUSIONS Lyar/p53 mutant mice represent a new digenic model of NTDs. Furthermore, these studies identify Lyar as a novel candidate gene for a role in human NTDs. These results provide new data to support the idea that loss of a p53-mediated developmental checkpoint may increase the risk of NTDs owing to some germline mutations.

Targeting mitochondrial translation by inhibiting DDX3: a novel radiosensitization strategy for cancer treatment

DDX3 is a DEAD box RNA helicase with oncogenic properties. RK-33 is developed as a small-molecule inhibitor of DDX3 and showed potent radiosensitizing activity in preclinical tumor models. This study aimed to assess DDX3 as a target in breast cancer and to elucidate how RK-33 exerts its anti-neoplastic effects. High DDX3 expression was present in 35% of breast cancer patient samples and correlated with markers of aggressiveness and shorter survival. With a quantitative proteomics approach, we identified proteins involved in the mitochondrial translation and respiratory electron transport pathways to be significantly downregulated after RK-33 or DDX3 knockdown. DDX3 localized to the mitochondria and DDX3 inhibition with RK-33 reduced mitochondrial translation. As a consequence, oxygen consumption rates and intracellular ATP concentrations decreased and reactive oxygen species (ROS) increased. RK-33 antagonized the increase in oxygen consumption and ATP production observed after exposure to ionizing radiation and reduced DNA repair. Overall, we conclude that DDX3 inhibition with RK-33 causes radiosensitization in breast cancer through inhibition of mitochondrial translation, which results in reduced oxidative phosphorylation capacity and increased ROS levels, culminating in a bioenergetic catastrophe.