Inhibition of NOTCH4 sensitizes FLT3/ITD acute myeloid leukemia cells to FLT3 tyrosine kinase inhibition.

Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition. Further investigation found increased NOTCH4 expression in FLT3/ITD AML cell lines and primary patient samples. Inhibition of NOTCH4 by shRNA knockdown, CRISPR-Cas9-based knockout or γ-secretase inhibitors synergized with FLT3 TKIs to kill FLT3/ITD AML cells in vitro. NOTCH4 inhibition sensitized TKI-resistant FLT3/ITD cells to FLT3 TKI inhibition. The combination reduced phospho-ERK and phospho-AKT, indicating inhibition of MAPK and PI3K/AKT signaling pathways. It also led to changes in expression of genes involved in regulating cell cycling, DNA repair and transcription. A patient-derived xenograft model showed that the combination reduced both the level of leukemic involvement of primary human FLT3/ITD AML cells and their ability to engraft secondary recipients. In summary, these results demonstrate that NOTCH4 inhibition synergizes with FLT3 TKIs to eliminate FLT3/ITD AML cells, providing a new therapeutic target for AML with FLT3/ITD mutations.

All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo.

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)(+) LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD(+) cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD(+) cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD(+) LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD(+) patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drug's apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD(+) LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.

HHV-8-positive and EBV-positive intravascular lymphoma: an unusual presentation of extracavitary primary effusion lymphoma.

Intravascular lymphomas are rare and aggressive hematolymphoid tumors. Here, we describe a human herpesvirus type-8 (HHV-8)/Kaposi sarcoma-associated herpesvirus-positive and Epstein-Barr virus (EBV)-positive intravascular lymphoma. The patient was a 59-year-old human immunodeficiency virus-positive man who presented with diarrhea, abdominal pain, fevers, night sweats, and weight loss. Radiographic studies of the abdomen and pelvis revealed numerous subcentimeter nodules within the subcutaneous fat that lacked connection to the skin. An excisional biopsy demonstrated large atypical cells within vessels in the deep subcutaneous fat, and many of the vessels contained extensive organizing thrombi. The atypical cells lacked strong expression of most B-cell markers but were positive for MUM-1 and showed partial expression of several T-cell markers. An immunohistochemical stain for HHV-8 and an in situ hybridization for EBV were both positive in the neoplastic cells. The disease had a rapidly progressive and fatal course. This lymphoma appears to represent an entirely intravascular form of primary effusion lymphoma and highlights the propensity for HHV-8 and EBV-positive lymphoid neoplasms to show aberrant expression of T-cell markers, illustrates the utility of skin biopsies for the diagnosis of intravascular lymphoma, and suggests that biopsies to evaluate for intravascular lymphoma should be relatively deep and include subcutaneous fat.

Nelfinavir inhibits maturation and export of herpes simplex virus 1.

UNLABELLED: Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment. We confirmed the inhibitory effects of NFV on HSV-1 replication by plaque assay and found that treatment with NFV did not affect capsid assembly, activity of the HSV-1 maturational protease, or formation of DNA-containing capsids in the nucleus. Confocal and electron microscopy studies showed that these capsids were transported to the cytoplasm but failed to complete secondary envelopment and subsequent exit from the cell. Consistent with the microscopy results, a light-scattering band corresponding to enveloped virions was not evident following sucrose gradient rate-velocity separation of lysates from drug-treated cells. Evidence of a possibly related effect of NFV on viral glycoprotein maturation was also discovered. NFV also inhibited the replication of an HSV-1 thymidine kinase mutant resistant to nucleoside analogues such as acyclovir. Given that NFV is neither a nucleoside mimic nor a known inhibitor of nucleic acid synthesis, this was expected and suggests its potential as a coinhibitor or alternate antiviral therapeutic agent in cases of resistance. IMPORTANCE: Nelfinavir (NFV) is a clinically important antiviral drug that inhibits production of infectious HIV. It was reported to inhibit herpesviruses in cell culture. Herpes simplex virus 1 (HSV-1) infections are common and often associated with several diseases. The studies we describe here confirm and extend earlier findings by investigating how NFV interferes with HSV-1 replication. We show that early steps in virus formation (e.g., assembly of DNA-containing capsids in the nucleus and their movement into the cytoplasm) appear to be unaffected by NFV, whereas later steps (e.g., final envelopment in the cytoplasm and release of infectious virus from the cell) are severely restricted by the drug. Our findings provide the first insight into how NFV inhibits HSV-1 replication and suggest that this drug may have applications for studying the herpesvirus envelopment process. Additionally, NFV may have therapeutic value alone or in combination with other antivirals in treating herpesvirus infections.

When differentiation goes viral.

In this issue of Blood, Vrzalikova et al present evidence that Epstein-Barr virus (EBV) infection leads to the down-regulation of BLIMP1 in primary germinal center (GC) B cells. BLIMP1 is a transcription factor encoded by the PRDM1 gene. It stops cell cycling and leads to terminal differentiation of B cells to plasma cells. The authors suggest that EBV inhibition of BLIMP1 expression serves to inhibit terminal differentiation of EBV-infected cells.

Bortezomib induction of C/EBPß mediates Epstein-Barr virus lytic activation in Burkitt lymphoma.

Epstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinß (C/EBPß) in a variety of tumor cell lines. C/EBPß activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPß inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPß knock-down. Therefore, C/EBPß mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.