HIV-1 gp120 Impairs Spatial Memory Through Cyclic AMP Response Element-Binding Protein.

HIV-associated neurocognitive disorders (HAND) remain an unsolved problem that persists despite using antiretroviral therapy. We have obtained data showing that HIV-gp120 protein contributes to neurodegeneration through metabolic reprogramming. This led to decreased ATP levels, lower mitochondrial DNA copy numbers, and loss of mitochondria cristae, all-important for mitochondrial biogenesis. gp120 protein also disrupted mitochondrial movement and synaptic plasticity. Searching for the mechanisms involved, we found that gp120 alters the cyclic AMP response element-binding protein (CREB) phosphorylation on serine residue 133 necessary for its function as a transcription factor. Since CREB regulates the promoters of PGC1α and BDNF genes, we found that CREB dephosphorylation causes PGC1α and BDNF loss of functions. The data was validated in vitro and in vivo. The negative effect of gp120 was alleviated in cells and animals in the presence of rolipram, an inhibitor of phosphodiesterase protein 4 (PDE4), restoring CREB phosphorylation. We concluded that HIV-gp120 protein contributes to HAND via inhibition of CREB protein function.

Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies.

Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. SIGNIFICANCE: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.

Combinatorial efficacy of entospletinib and chemotherapy in patient-derived xenograft models of infant acute lymphoblastic leukemia.

Survival of infants with KMT2A-rearranged (R) acute lymphoblastic leukemia (ALL) remains dismal despite intensive chemotherapy. We observed constitutive phosphorylation of spleen tyrosine kinase (SYK) and associated signaling proteins in infant ALL patient-derived xenograft (PDX) model specimens and hypothesized that the SYK inhibitor entospletinib would inhibit signaling and cell growth in vitro and leukemia proliferation in vivo. We further predicted that combined entospletinib and chemotherapy could augment anti-leukemia effects. Basal kinase signaling activation and HOXA9/MEIS1 expression differed among KMT2A-R (KMT2A-AFF1 [n=4], KMT2A-MLLT3 [n=1], KMT2A-MLLT1 [n=4]) and non-KMT2A-R [n=3] ALL specimens and stratified by genetic subgroup. Incubation of KMT2A-R ALL cells in vitro with entospletinib inhibited methylcellulose colony formation and SYK pathway signaling in a dose-dependent manner. In vivo inhibition of leukemia proliferation with entospletinib monotherapy was observed in RAS-wild-type KMT2A-AFF1, KMT2A-MLLT3, and KMT2A-MLLT1 ALL PDX models with enhanced activity in combination with vincristine chemotherapy in several models. Surprisingly, entospletinib did not decrease leukemia burden in two KMT2A-AFF1 PDX models with NRAS/ or KRAS mutations, suggesting potential RAS-mediated resistance to SYK inhibition. As hypothesized, superior inhibition of ALL proliferation was observed in KMT2A-AFF1 PDX models treated with entospletinib and the MEK inhibitor selumetinib versus vehicle or inhibitor monotherapies (p.

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL.

Children and adults with Philadelphia chromosome-like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor-like factor 2-rearranged (CRLF2-rearranged) Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe an adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK, and PI3K signaling consistent with activated B cell receptor (BCR) signaling, although they do not express cell surface µ-heavy chain (µHC). Combinatorial targeting of JAK/STAT, PI3K, and "BCR-like" signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.

CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy.

Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19- subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)- deep remission, whereas 67 patients had a recurrence after achieving a MRD- deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19- leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19- leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19- events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD- remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

HIV-1 Tat protein promotes neuronal dysregulation by inhibiting E2F transcription factor 3 (E2F3).

Individuals who are infected with HIV-1 accumulate damage to cells and tissues (e.g. neurons) that are not directly infected by the virus. These include changes known as HIV-associated neurodegenerative disorder (HAND), leading to the loss of neuronal functions, including synaptic long-term potentiation (LTP). Several mechanisms have been proposed for HAND, including direct effects of viral proteins such as the Tat protein. Searching for the mechanisms involved, we found here that HIV-1 Tat inhibits E2F transcription factor 3 (E2F3), CAMP-responsive element-binding protein (CREB), and brain-derived neurotropic factor (BDNF) by up-regulating the microRNA miR-34a. These changes rendered murine neurons dysfunctional by promoting neurite retraction, and we also demonstrate that E2F3 is a specific target of miR-34a. Interestingly, bioinformatics analysis revealed the presence of an E2F3-binding site within the CREB promoter, which we validated with ChIP and transient transfection assays. Of note, luciferase reporter assays revealed that E2F3 up-regulates CREB expression and that Tat interferes with this up-regulation. Further, we show that miR-34a inhibition or E2F3 overexpression neutralizes Tat's effects and restores normal distribution of the synaptic protein synaptophysin, confirming that Tat alters these factors, leading to neurite retraction inhibition. Our results suggest that E2F3 is a key player in neuronal functions and may represent a good target for preventing the development of HAND.

Aberrant splicing in B-cell acute lymphoblastic leukemia.

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ∼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.

CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum.

We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

UNLABELLED: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Involvement of miR-196a in HIV-associated neurocognitive disorders.

Involvement of the human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) protein in neuronal deregulation and in the development of HIV-1 associated neurocognitive disorders (HAND) has been amply explored; however the mechanisms involved remain unclear. In search for the mechanisms, we demonstrated that Tat deregulates neuronal functions through a pathway that involved p73 and p53 pathway. We showed that Tat uses microRNA-196a (miR-196a) to deregulate the p73 pathway. Further, we found that the Abelson murine leukemia (c-Abl) phosphorylates p73 on tyrosine residue 99 (Tyr-99) in Tat-treated cells. Interestingly, Tat lost its ability to promote accumulation and phosphorylation of p73 in the presence of miR-196a mimic. Interestingly, accumulation of p73 did not lead to neuronal cell death by apoptosis as obtained by cell viability assay. Western blot analysis using antibodies directed against serine residues 807 and 811 of retinoblastoma (Rb) protein was also used to validate our data regarding lack of cell death. Hyperphosphorylation of RB (S807/811) is an indication of cell neuronal viability. These results highlight the key role played by p73 and microRNA in Tat-treated neurons leading to their deregulation and it deciphers mechanistically one of the pathways used by Tat to cause neuronal dysfunction that contributes to the development of HAND.

Noncoding RNAs and LRRFIP1 regulate TNF expression.

Noncoding RNAs have been implicated in the regulation of expression of numerous genes; however, the mechanism is not fully understood. We identified bidirectional, long noncoding RNAs upstream of the TNF gene using five different methods. They arose in a region where the repressors LRRFIP1, EZH2, and SUZ12 were demonstrated to bind, suggesting a role in repression. The noncoding RNAs were polyadenylated, capped, and chromatin associated. Knockdown of the noncoding RNAs was associated with derepression of TNF mRNA and diminished binding of LRRFIP1 to both RNA targets and chromatin. Overexpression of the noncoding RNAs led to diminished expression of TNF and recruitment of repressor proteins to the locus. One repressor protein, LRRFIP1, bound directly to the noncoding RNAs. These data place the noncoding RNAs upstream of TNF gene as central to the transcriptional regulation. They appear to serve as a platform for the assembly of a repressive complex.

Roles and functions of HIV-1 Tat protein in the CNS: an overview.

Nearly 50% of HIV-infected individuals suffer from some form of HIV-associated neurocognitive disorders (HAND). HIV-1 Tat (a key HIV transactivator of transcription) protein is one of the first HIV proteins to be expressed after infection occurs and is absolutely required for the initiation of the HIV genome transcription. In addition to its canonical functions, various studies have shown the deleterious role of HIV-1 Tat in the development and progression of HAND. Within the CNS, only specific cell types can support productive viral replication (astrocytes and microglia), however Tat protein can be released form infected cells to affects HIV non-permissive cells such as neurons. Therefore, in this review, we will summarize the functions of HIV-1 Tat proteins in neural cells and its ability to promote HAND.

Cdk9 phosphorylates Pirh2 protein and prevents degradation of p53 protein.

Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long-terminal repeat (HIV-1 LTR). We recently demonstrated that through their physical interaction, cdk9 phosphorylates p53 on Ser-392, leading to p53 stability and accumulation. As a result, p53 stalled transcriptional elongation of the HIV-1 LTR and significantly reduced HIV-1 replication in primary microglia and astrocytes. Therefore, we sought to identify the mechanisms used by cdk9 to allow this p53 function. Using western blot analysis, we found that cdk9 promotes inhibition and phosphorylation of Mdm2 on Ser-395, thus preventing degradation of p53, a protein that is directly involved in promoting p53 ubiquitination. On the other hand, we showed that cdk9 phosphorylates Pirh2 on Ser-211 and Thr-217 residues through their physical interaction. Phosphorylation of Pirh2 renders it inactive and may contribute to p53-inhibition of transcriptional elongation of the HIV-1 LTR. Hence, we suggest that phosphorylation of Pirh2 may be a novel target for the inhibition of HIV-1 gene expression.

Role of p53 in neurodegenerative diseases.

BACKGROUND: p53 plays an important role in many areas of cellular physiology and biology, ranging from cellular development and differentiation to cell cycle arrest and apoptosis. Many of its functions are attributed to its role in assuring proper cellular division. However, since the establishment of its role in cell cycle arrest, damage repair, and apoptosis (thus also establishing its importance in cancer development), numerous reports have demonstrated additional functions of p53 in various cells. In particular, p53 appears to have important functions as it relates to neurodegeneration and synaptic plasticity. OBJECTIVE: In this review, we will address p53 functions as it relates to various neurodegenerative diseases, mainly its implications in the development of HIV-associated neurocognitive disorders. CONCLUSION: p53 plays a pivotal role in the development of neurodegenerative diseases through its interaction with cellular factors, viral factors, and/or small RNAs that have the ability to promote the development of these diseases. Hence, inhibition of p53 may present an ideal target to restore neuronal functions.

HIV-1 Tat protein promotes neuronal dysfunction through disruption of microRNAs.

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders.

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.

Characterization of LRRFIP1.

LRRFIP1 has been identified as a regulator of toll-like receptor (TLR) pathway signaling; however, little is known about its own regulation and function. This study was undertaken to characterize the biochemical properties and its regulation. Over-expression of full length LRRFIP1 led to enhanced responses to lipopolysaccharide (LPS). We examined its expression in monocytic cell lines because they express a broad range of TLRs. We found that its level of expression was not altered by LPS or phorbol myristate acetate (PMA) but that it was up-regulated by nicotine, influenza infection, and serum starvation. Phosphorylation was examined because of the bioinformatically predicted serine phosphorylation sites. Serine phosphorylation was detected and was altered by both poly I:C and nicotine. Finally, we examined the regulation of intracellular localization in response to dsRNA and found that LRRFIP1 colocalized with labeled dsRNA in monocyte lysosomal structures but not with lysosomes lacking dsRNA. These data suggest that LRRFIP1 is phosphorylated in response to immunologic stimuli and it is directed to lysosomal structures.