HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

HDAC11 activity contributes to MEK inhibitor escape in uveal melanoma.

We previously demonstrated that pan-HDAC inhibitors could limit escape from MEK inhibitor (MEKi) therapy in uveal melanoma (UM) through suppression of AKT and YAP/TAZ signaling. Here, we focused on the role of specific HDACs in therapy adaptation. Class 2 UM displayed higher expression of HDACs 1, 2, and 3 than Class 1, whereas HDACs 6, 8, and 11 were uniformly expressed. Treatment of UM cells with MEKi led to modulation of multiple HDACs, with the strongest increases observed in HDAC11. RNA-seq analysis showed MEKi to decrease the expression of multiple HDAC11 target genes. Silencing of HDAC11 significantly reduced protein deacetylation, enhanced the apoptotic response to MEKi and reduced growth in long-term colony formation assays across multiple UM cell lines. Knockdown of HDAC11 led to decreased expression of TAZ in some UM cell lines, accompanied by decreased YAP/TAZ transcriptional activity and reduced expression of multiple YAP/TAZ target genes. Further studies showed this decrease in TAZ expression to be associated with increased LKB1 activation and modulation of glycolysis. In an in vivo model of uveal melanoma, silencing of HDAC11 limited the escape to MEKi therapy, an effect associated with reduced levels of Ki67 staining and increased cleaved caspase-3. We have demonstrated a novel role for adaptive HDAC11 activity in UM cells, that in some cases modulates YAP/TAZ signaling leading to MEKi escape.

Ironing-Out the Details: New Strategies for Combining Ferroptosis Inhibitors with Immunotherapy in Melanoma.

Escape from ferroptosis is an important determinant of metastasis and immune evasion in melanoma. In a new article of the Journal of Investigative Dermatology, Wang et al. (2021) identify the CAMKK2‒adenosine monophosphate-activated protein kinase‒NRF2 signaling axis as a negative regulator of ferroptosis and showed that inhibiting CAMKK2 increases the efficacy of anti-PD-1 therapy. These findings offer new opportunities for the development of ferroptosis-inducing therapies to use in combination with immune checkpoint agents.

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma.

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. In vivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.

Decitabine limits escape from MEK inhibition in uveal melanoma.

MEK inhibitors (MEKi) demonstrate anti-proliferative activity in patients with metastatic uveal melanoma, but responses are short-lived. In the present study, we evaluated the MEKi trametinib alone and in combination with drugs targeting epigenetic regulators, including DOT1L, EZH2, LSD1, DNA methyltransferases, and histone acetyltransferases. The DNA methyltransferase inhibitor (DNMTi) decitabine effectively enhanced the anti-proliferative activity of trametinib in cell viability, colony formation, and 3D organoid assays. RNA-Seq analysis showed the MEKi-DNMTi combination primarily affected the expression of genes involved in G1 and G2/2M checkpoints, cell survival, chromosome segregation and mitotic spindle. The DNMTi-MEKi combination did not appear to induce a DNA damage response (as measured by γH2AX foci) or senescence (as measured by ß-galactosidase staining) compared to either MEKi or DNMTi alone. Instead, the combination increased expression of the CDK inhibitor p21 and the pro-apoptotic protein BIM. In vivo, the DNMTi-MEKi combination was more effective at suppressing growth of MP41 uveal melanoma xenografts than either drug alone. Our studies indicate that DNMTi may enhance the activity of MEKi in uveal melanoma.

HDAC Inhibition Enhances the In Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma.

PURPOSE: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance.Experimental Design: Mass spectrometry-based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma. RESULTS: We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3-mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling. CONCLUSIONS: Together, our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.

HDAC8 Regulates a Stress Response Pathway in Melanoma to Mediate Escape from BRAF Inhibitor Therapy.

Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug-resistant phenotype. Mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF-MEK inhibition. SIGNIFICANCE: This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2947/F1.large.jpg.

MTI-101 treatment inducing activation of Stim1 and TRPC1 expression is a determinant of response in multiple myeloma.

The emergence of drug resistance continues to be a major hurdle towards improving patient outcomes for the treatment of Multiple Myeloma. MTI-101 is a first-in-class peptidomimetic that binds a CD44/ITGA4 containing complex and triggers necrotic cell death in multiple myeloma cell lines. In this report, we show that acquisition of resistance to MTI-101 correlates with changes in expression of genes predicted to attenuate Ca2+ flux. Consistent with the acquired resistant genotype, MTI-101 treatment induces a rapid and robust increase in intracellular Ca2+ levels in the parental cells; a finding that was attenuated in the acquired drug resistant cell line. Mechanistically, we show that pharmacological inhibition of store operated channels or reduction in the expression of a component of the store operated Ca2+ channel, TRPC1 blocks MTI-101 induced cell death. Importantly, MTI-101 is more potent in specimens obtained from relapsed myeloma patients, suggesting that relapse may occur at a cost for increased sensitivity to Ca2+ overload mediated cell death. Finally, we demonstrate that MTI-101 is synergistic when combined with bortezomib, using both myeloma cell lines and primary myeloma patient specimens. Together, these data continue to support the development of this novel class of compounds for the treatment of relapsed myeloma.

The role of phenotypic plasticity in the escape of cancer cells from targeted therapy.

Targeted therapy has proven to be beneficial at producing significant responses in patients with a wide variety of cancers. Despite initially impressive responses, most individuals ultimately fail these therapies and show signs of drug resistance. Very few patients are ever cured. Emerging evidence suggests that treatment of cancer cells with kinase inhibitors leads a minor population of cells to undergo a phenotypic switch to a more embryonic-like state. The adoption of this state, which is analogous to an epithelial-to-mesenchymal transition, is associated with drug resistance and increased tumor aggressiveness. In this commentary we will provide a comprehensive analysis of the mechanisms that underlie the embryonic reversion that occurs on targeted cancer therapy and will review potential novel therapeutic strategies designed to eradicate the escaping cells.

CRM1 Inhibition Sensitizes Drug Resistant Human Myeloma Cells to Topoisomerase II and Proteasome Inhibitors both In Vitro and Ex Vivo.

Multiple myeloma (MM) remains an incurable disease despite improved treatments, including lenalidomide/pomalidomide and bortezomib/carfilzomib based therapies and high-dose chemotherapy with autologous stem cell rescue. New drug targets are needed to further improve treatment outcomes. Nuclear export of macromolecules is misregulated in many cancers, including in hematological malignancies such as MM. CRM1 (chromosome maintenance protein-1) is a ubiquitous protein that exports large proteins (>40 kDa) from the nucleus to the cytoplasm. We found that small-molecule Selective Inhibitors of Nuclear Export (SINE) prevent CRM1-mediated export of p53 and topoisomerase IIα (topo IIα). SINE's CRM1-inhibiting activity was verified by nuclear-cytoplasmic fractionation and immunocytochemical staining of the CRM1 cargoes p53 and topo IIα in MM cells. We found that SINE molecules reduced cell viability and induced apoptosis when used as both single agents in the sub-micromolar range and when combined with doxorubicin, bortezomib, or carfilzomib but not lenalidomide, melphalan, or dexamethasone. In addition, CRM1 inhibition sensitized MM cell lines and patient myeloma cells to doxorubicin, bortezomib, and carfilzomib but did not affect peripheral blood mononuclear or non-myeloma bone marrow mononuclear cells as shown by cell viability and apoptosis assay. Drug resistance induced by co-culture of myeloma cells with bone marrow stroma cells was circumvented by the addition of SINE molecules. These results support the continued development of SINE for patients with MM.

MTI-101 (cyclized HYD1) binds a CD44 containing complex and induces necrotic cell death in multiple myeloma.

Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma cell lines. Because of the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels, and failed to activate caspase-3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and liquid chromatography/tandem mass spectrometry analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in multiple myeloma cell lines, indicating that multiple myeloma cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101-induced cell death. Mechanistically, we show that MTI-101-induced cell death occurs via a Rip1-, Rip3-, or Drp1-dependent and -independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma.

Emerging strategies for targeting cell adhesion in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological cancer involving proliferation of abnormal plasma cells that infiltrate the bone marrow (BM) and secrete monoclonal antibodies. The disease is clinically characterized by bone lesions, anemia, hypercalcemia, and renal failure. MM is presently treated with conventional therapies like melphalan, doxorubicin, and prednisone; or novel therapies like thalidomide, lenalidomide, and bortezomib; or with procedures like autologous stem cell transplantation. Unfortunately, these therapies fail to eliminate the minimal residual disease that remains persistent within the confines of the BM of MM patients. Mounting evidence indicates that components of the BM-including extracellular matrix, cytokines, chemokines, and growth factors-provide a sanctuary for subpopulations of MM. This co-dependent development of the disease in the context of the BM not only ensures the survival and growth of the plasma cells but contributes to de novo drug resistance. In addition, by fostering homing, angiogenesis, and osteolysis, this crosstalk plays a critical role in the progression of the disease. Not surprisingly then, over the past decade, several strategies have been developed to disrupt this communication between the plasma cells and the BM components including antibodies, peptides, and inhibitors of signaling pathways. Ultimately, the goal is to use these therapies in combination with the existing antimyeloma agents in order to further reduce or abolish minimal residual disease and improve patient outcomes.

Acquisition of resistance toward HYD1 correlates with a reduction in cleaved α4 integrin expression and a compromised CAM-DR phenotype.

We recently reported that the ß1 integrin antagonist, referred to as HYD1, induces necrotic cell death in myeloma cell lines as a single agent using in vitro and in vivo models. In this article, we sought to delineate the determinants of sensitivity and resistance toward HYD1-induced cell death. To this end, we developed an HYD1 isogenic resistant myeloma cell line by chronically exposing H929 myeloma cells to increasing concentrations of HYD1. Our data indicate that the acquisition of resistance toward HYD1 correlates with reduced levels of the cleaved α4 integrin subunit. Consistent with reduced VLA-4 (α4ß1) expression, the resistant variant showed ablated functional binding to fibronectin, VCAM-1, and the bone marrow stroma cell line HS-5. The reduction in binding of the resistant cell line to HS-5 cells translated to a compromised cell adhesion-mediated drug resistant phenotype as shown by increased sensitivity to melphalan- and bortezomib-induced cell death in the bone marrow stroma coculture model of drug resistance. Importantly, we show that HYD1 is more potent in relapsed myeloma specimens than newly diagnosed patients, a finding that correlated with α4 integrin expression. Collectively, these data indicate that this novel d-amino acid peptide may represent a good candidate for pursuing clinical trials in relapsed myeloma and in particular patients with high levels of α4 integrin. Moreover, our data provide further rationale for continued preclinical development of HYD1 and analogues of HYD1 for the treatment of multiple myeloma and potentially other tumors that home and/or metastasize to the bone.

Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition.

Topoisomerase IIalpha (topo IIalpha) is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism at cellular densities similar to those found in patient bone marrow. When topo IIalpha is trafficked to the cytoplasm, it is not in contact with the DNA; thus, topo IIalpha inhibitors are unable to induce DNA-cleavable complexes and cell death. Using a CRM1 inhibitor or a CRM1-specific small interfering RNA (siRNA), we were able to block nuclear export of topo IIalpha as shown by immunofluorescence microscopy. Human myeloma cell lines and patient myeloma cells isolated from bone marrow were treated with a CRM1 inhibitor or CRM1-specific siRNA and exposed to doxorubicin or etoposide at high cell densities. CRM1-treated cell lines or myeloma patient cells were 4-fold more sensitive to topo II poisons as determined by an activated caspase assay. Normal cells were not significantly affected by CRM1-topo II inhibitor combination treatment. Cell death was correlated with increased DNA double-strand breaks as shown by the comet assay. Band depletion assays of CRM1 inhibitor-exposed myeloma cells showed increased topo IIalpha covalently bound to DNA. Topo IIalpha knockdown by a topo IIalpha-specific siRNA abrogated the CRM1-topo II therapy synergistic effect. These results suggest that blocking topo IIalpha nuclear export sensitizes myeloma cells to topo II inhibitors. This method of sensitizing myeloma cells suggests a new therapeutic approach to multiple myeloma.

HYD1-induced increase in reactive oxygen species leads to autophagy and necrotic cell death in multiple myeloma cells.

HYD1 is a D-amino acid peptide that was previously shown to inhibit adhesion of prostate cancer cells to the extracellular matrix. In this study, we show that in addition to inhibiting adhesion of multiple myeloma (MM) cells to fibronectin, HYD1 induces cell death in MM cells as a single agent. HYD1-induced cell death was necrotic in nature as shown by: (a) decrease in mitochondrial membrane potential (Deltapsi(m)), (b) loss of total cellular ATP, and (c) increase in reactive oxygen species (ROS) production. Moreover, HYD1 treatment does not result in apoptotic cell death because it did not trigger the activation of caspases or the release of apoptosis-inducing factor and endonuclease G from the mitochondria, nor did it induce double-stranded DNA breaks. HYD1 did initiate autophagy in cells; however, autophagy was found to be an adaptive response contributing to cell survival rather than the cause of cell death. We were further able to show that N-acetyl-L-cysteine, a thiol-containing free radical scavenger, partially protects MM cells from HYD1-induced death. Additionally, N-acetyl-L-cysteine blocked HYD1-induced as well as basal levels of autophagy, suggesting that ROS can potentially trigger both cell death and cell survival pathways. Taken together, our data describe an important role of ROS in HYD1-induced necrotic cell death in MM cells.

Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance.

Imatinib mesylate is a potent, molecularly targeted therapy against the oncogenic tyrosine kinase BCR-ABL. Although imatinib mesylate has considerable efficacy against chronic myeloid leukemia (CML), advanced-stage CML patients frequently become refractory to this agent. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrices, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib mesylate sensitivity, we used an in vitro bone marrow stroma model. Our data show culturing K562 cells, in bone marrow stroma-derived conditioned medium (CM), is sufficient to cause resistance to BCR-ABL inhibitors. Drug resistance correlated with increased pTyrStat3, whereas no increases in pTyrStat5 was noted. Moreover, resistance was associated with increased levels of the Stat3 target genes Bcl-xl, Mcl-1, and survivin. Finally, reducing Stat3 levels with small interfering RNA sensitized K562 cells cultured in CM to imatinib mesylate-induced cell death. Importantly, Stat3 dependency was specific for cells grown in CM, as reducing Stat3 levels in regular growth conditions had no effect on imatinib mesylate sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib mesylate resistance and provides preclinical rationale for using Stat3-inhibitors to increase the efficacy of imatinib mesylate within the context of the bone marrow microenvironment.