BCL-2 antagonist venetoclax combined with hypomethylating agents or low-dose cytarabine is a new standard of care for treatment-naive elderly or unfit AML patients. Despite the striking overall ...response of 70%, only 30% achieved MRD negative status with a short remission duration of 11.3 months. This highlights the need for new agents that can overcome venetoclax resistance. We exploited mitochondrial functional assay called BH3 profiling to identify predictors of clinical responses and to discover combination partners of venetoclax. BH3 profiling measures apoptotic priming of a cell (proximity to the apoptotic threshold) by measuring mitochondrial response to a standardized panel of pro-apoptotic BH3 oligopeptides. By using pretreatment myeloblasts of patients (N=16) from clinical trial of venetoclax and azacitidine, we found that response to HRK + MS1 peptides (infers BCL-XL and MCL-1 dependency, respectively) inversely correlates with the achievement of remission. The serial sampling on treatment revealed the emergence of differential patterns of anti-apoptotic dependency that correlated with patient response. Of note, mitochondrial sensitivity to the MS-1 peptide, an indication of MCL-1 dependence, increased from 0% at diagnosis to 40% at relapse, suggesting an opportunity for MCL-1 antagonism in the relapsed setting.
First, we exposed 6 AML cell lines with a BCL-2 (venetoclax) and MCL-1 antagonist (S63845). As previously reported, combination treatment resulted in synergistic cell line killing. Venetoclax treatment (within an hour) led to a dynamic increase in mitochondrial sensitivity to the MCL-1 selective MS-1 peptide, while S63845 caused increased mitochondrial susceptibility to the BCL-2 and BCL-XL selective BAD peptide. There was a correlation between % of priming caused by MS-1 and BAD peptide with Loewe synergy score (Spearman r=0.79, p<0.05). This suggests that BH3 profiling can provide a rapid pharmacodynamic marker of the activity of BH3 mimetics.
We next investigated combination MCL-1 and BCL-2 antagonism in vivo in venetoclax resistant AML PDXs. We transplanted NSG mice with human AML cells and started them on venetoclax (100mg/kg, PO) until leukemia relapsed (N=6 models). We subjected resistant myeloblasts to BH3 profiling and identified dependency on BCL-2 and MCL-1 to guide therapeutic choices. As a single agent, S63845 showed limited activity in venetoclax resistance models (p<0.01 vs. vehicle; N=2/4 models). We next tested combinations at 3 different dosing schedules where BH3 mimetics were given either in a simultaneous week, alternative weeks or switched based on leukemia burden. Mice receiving venetoclax and S63845 in the same week showed the greatest anti-leukemia effects (p<0.01 to p<0.0005; N=4/4 models). We found superior benefit of BCL-2 + MCL-1 combination antagonism over MCL-1 alone in venetoclax resistance settings, implies that resistant myeloblasts escape apoptosis by switching their anti-apoptotic dependencies.
Next, we applied the dynamic BH3 profiling technique that measures drug-induced changes in mitochondrial priming to discover additional agents that may overcome venetoclax resistance. We compared mitochondrial priming signals of 40 targeted agents in isolated parental and resistant cells. Although venetoclax resistant cells gained cross resistance to most of the agents, inhibitors of the FLT-3 pathway, including quizartinib, crenolanib, and gilteritinib retained similar priming responses as in parental cells. To validate this, we re-transplanted venetoclax resistant cells and found that quizartinib treatment maintained anti-leukemia efficacy compared to venetoclax or vehicle-treated mice (p<0.0001). In an unbiased whole transcriptome analysis, venetoclax resistant PDXs showed enrichment for JAK/STAT, RAS/MAPK, and PI3K/AKT/mTOR pathways, and corresponding upregulation in protein levels of p-FLT3, p-STAT5, p-MAPK, and p-AKT. In addition we identified that FLT3-ITD mutation measured via capillary electrophoresis was maintained in resistant clones while point mutation in BCL2 (BCL2G101V) was undetectable.
In conclusion, our study illustrates the power of mitochondrial measurements as a predictive biomarker for BH3 mimetic based therapy. We provide an evidence for the persistent activity of FLT-3 inhibitors in venetoclax resistance settings, a discovery made possible by dynamic BH3 profiling.
Ryan:Vivid Biosciences: Consultancy. Erick:Novartis: Employment. Weinstock:Celgene: Research Funding. Garcia:Abbvie: Research Funding; Genentech: Research Funding. Letai:Novartis: Research Funding; Flash Therapeutics: Equity Ownership; Abbvie: Consultancy, Research Funding; Vivid Bioscience: Equity Ownership; Astrazeneca: Research Funding.
Background
Acute myeloid leukemia (AML) cells harboring mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) produce the oncometabolite 2‐hydroxyglutarate (2HG). This ...study prospectively evaluated the 2HG levels, IDH1/2 mutational status, and outcomes of patients receiving standard chemotherapy for newly diagnosed AML.
Methods
Serial samples of serum, urine, and bone marrow aspirates were collected from patients newly diagnosed with AML, and 2HG levels were measured with mass spectrometry. Patients with baseline serum 2HG levels greater than 1000 ng/mL or marrow pellet 2HG levels greater than 1000 ng/2 × 106 cells, which suggested the presence of an IDH1/2 mutation, underwent serial testing. IDH1/2 mutations and estimated variant allele frequencies were identified. AML characteristics were compared with the Wilcoxon test and Fisher’s exact test. Disease‐free survival and overall survival (OS) were evaluated with log‐rank tests and Cox regression.
Results
Two hundred and two patients were treated for AML; 51 harbored IDH1/2 mutations. IDH1/2‐mutated patients had significantly higher 2HG levels in serum, urine, bone marrow aspirates, and aspirate cell pellets than wild‐type patients. A serum 2HG level greater than 534.5 ng/mL was 98.8% specific for the presence of an IDH1/2 mutation. Patients with IDH1/2‐mutated AML treated with 7+3‐based induction had a 2‐year event‐free survival (EFS) rate of 44% and a 2‐year OS rate of 57%. There was no difference in complete remission rates, EFS, or OS between IDH1/2‐mutated and wild‐type patients. Decreased serum 2HG levels on day 14 as a proportion of the baseline were significantly associated with improvements in EFS (P = .047) and OS (P = .019) in a multivariate analysis.
Conclusions
Among patients with IDH1/2‐mutated AML, 2HG levels are highly specific for the mutational status at diagnosis, and they have prognostic relevance in patients receiving standard chemotherapy.
In this prospective study, isocitrate dehydrogenase mutated patients have higher 2‐hydroxyglutarate levels in serum, urine, and marrow than wild‐type patients, whereas decreased serum 2‐hydroxyglutarate levels on day 14 of treatment, as a proportion of the baseline, are associated with better survival. In isocitrate dehydrogenase mutated acute myeloid leukemia, 2‐hydroxyglutarate levels are highly specific for the mutational status at diagnosis and have a prognostic impact on patients who are receiving conventional treatments.
RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis; both pathways are undruggable. We evaluated molecular changes associated with pathway-level responses after ...RAS/CDK inhibition to identify novel molecular targets.
in our previous studies MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to target RAS/CDK pathways. Our studies indicate strong synergistic (IC<0.5) MM cytotoxicity triggered by Ei+Ci treatment, which in a dose-dependent manner arrested MM cells in G0/G1 phase and activated mitochondrial apoptotic signaling. Ei+Ci treatment decreased Erk1/2, CDK4/6, and p-Erk1/2 levels in MM cells, and also induced inhibition of key targets (c-myc, p-RSK,-RB, E2F1) of RAS/CDK cascade. Our studies in patient samples indicate that MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that Ei+Ci combination can overcome the protective effects of the MM BM milieu. An in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. Our studies therefore suggesting on-target activity of these inhibitors in vitro/vivo.
We evaluated mRNA splicing changes in MM cells, with and without Erk1/2 knockdown or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. Upregulation in response to Erk1/2 knockdown and downregulation due to treatment with Ei+Ci were considered spliced gene signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complexes and are involved in intron retention processes, a marker of malignant transformation. We evaluated expressions of RAVER1 and SNRPB in 558 MM patent samples and 10 normal donor BM PCs and observed significant (p<2e-11) upregulation of both genes in clonal PCs with progression from MGUS to sMM, and to overt MM. SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 has a trend toward poor outcomes. SNRPB proteins are also overexpressed in MM cells. Evaluating SNRPB effects on RNA splicing showed upregulation of transcripts with full intron retention or transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. Thus, SNRPB overexpression contributes to aberrant transcriptome splicing associated with RAS/CDK cascade in MM.
Our studies show an association between RNA processing and RAS-CDK pathways in MM, identify a core splicing protein, SNRPB, as a novel target for modulating this undruggable cascade, and suggest that targeting spliceosome complexes is a promising therapy.
Mutations in two type-3 receptor tyrosine kinases (RTK), KIT and FLT3, are common in both acute myeloid leukemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signaling ...pathways. Fortunately, a significant number of tyrosine kinase inhibitors TKIs have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been FDA approved for mutant FLT3-positive AML and for KIT-D816V-positive SM. Here, we compare the
in vitro
activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-ITD, FLT3-D835Y, the resistance mutant FLT3-ITD+F691L, KIT-D816V, and KIT-N822K. We also examined the effects of these inhibitors
in vitro
and
in vivo
on cells expressing mutations in c-CBL found in AML that result in hypersensitization of TK receptors such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.
The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have antileukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is ...frequently limited. The success of current antileukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here, we evaluated the antileukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents.
The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n = 6; B-CLL, n = 19), and healthy leukocytes. Intracellular nicotinamide adenine dinucleotide (NAD(+)) and ATP levels, mitochondrial transmembrane potential (ΔΨ(m)), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated.
The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34(+) progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD(+) and ATP shortage, and induced ΔΨ(m) dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments.
APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including acute myelogenous leukemia (AML) and B-cell chronic lymphocytic leukemia (B-CLL) samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Genome-wide transcriptome profiling detected an increased splicing alterations in MM and AML. While these malignancies are derived from different cell linages, their tumor cells acquire similar ...aberrant splicing (AbSp), mostly intron retentions. To delineate AbSp mechanism in MM/AML, we focused on PTBPs (1/2/3) that play a critical role in intron excision. We have previously reported deregulated expression of splicing factors (SFs) in MM/AML patient and healthy donor (HD) bone marrow (BM). As MM progressed, PTBP1/2 progressively increased, and PTBP3 gradually decreased (ASH 2017). In AML, PTBP2/3 upregulation and PTBP1 downregulation were detected in patient samples in which increased intron retentions were identified by genome-wide splicing analysis (CCR 2015).
Here, these findings were validated by TaqMan assays for PTBPs in 48 MM and 325 AML patient samples, 16 MM/AML cell lines, and in plasma cells (PCs) and CD34+cells from 14 HDBM. Results were consistent with differential expressions of PTBPs in MM/AML previously analyzed. Upregulation of PTBP1/2 and PTBP2/3 proteins were detected in MM and AML cell lines, respectively. PTBP1/2 upregulation was pronounced when MM cell lines were cocultured with BM stromal cells (MMBMSC) derived from MM patients' BM. Importantly, we detected increased proliferation and decreased apoptosis in MM/AML cell lines overexpressing PTBPs. These effects were most evident after coculturing MM cell lines with MMBMSC as compared to HDBMSC.
To evaluate PTBP effects on AbSp, we knocked down and/or overexpressed PTBPs in MM/AML cell lines and assessed PDL1 splicing in MM, and NOTCH2 and FLT3 splicing in AML. PDL1 is spliced in ~ 30% of 90 MM patients; while NOTCH2/FLT3/CD13 are spliced in 78%/50% of 387 AML patients, respectively. After PTBP1/2 knockdown in MM cells we detected 4- to 11-fold downregulation of PDL1 splice variants, with proportional upregulation of wild-type PDL1 levels; concurrently, MM cell proliferation was decreased and apoptosis increased. To evaluate MM specific splicing alterations in the context of the MM BM microenvironment, PDL1 splicing, and PTBP1/2 mRNA/protein expressions, were monitored in MM cell lines cocultured with MMBMSC or HDBMSC. We observed time-dependent PDL1 splice variant upregulation, and higher levels of PTBP1/2 in MM cells. We also noted time-dependent PDL1 variant expression switching in association with PTBP1/2 deregulation in the BM microenvironment. RNA-seq analysis and western blotting showed that MMBMSC culture with tumor cells increased intron retention, and altered SF expressions, including PTBPs in BMSC.
We next monitored PTBP effects on splicing in AML cells by evaluating NOTCH2 and FLT3 splicing in an TF1, an AML cell line that overexpresses PTBP2/3. RT-PCR analysis showed association between PTBP3 overexpression and NOTCH2 and FLT3 AbSp in TF1 cells. For further validation, we developed an ex vivo splicing assay composed of an FLT3/CD13 splicing cassette with a GFP reporter, which allows for evaluation of splicing events by flow cytometry (FACS) and microscopy. In this assay, cells overexpressing PTBP2/3 caused FLT3/CD13 minigene splicing similar to that detected in AML patients. Also, by RT-PCR, we showed that overexpression of PTBPs caused intron retention, that was confirmed by cloning and sequencing of PCR products and consistent with the FACS analysis and microscopy.
Finally, we have tested effects of PTBPs using in vivo AML models (BMT and xenograft). In the BMT model, animal median survival was 48 days post-BMT for MLL-AF9/PTBP3 and 56 days in the control group. In the xenograft model, animal median survival was 66, 94, & 106 days after injection of TF1-PTBP3, TF1-PTBP2, and TF1 cells in mice, respectively (P>0.0001). These studies suggest that PTBP3 overexpression in partnership with the MLL-AF9 promotes occurrence of AML in mice. Tumor RNA samples harvested from these animals were subjected to RNA-seq analysis, which showed increased AbSp association with PTBP3 overexpression.
Our studies indicate that deregulated PTBP1/2 expression in MM and of PTBP2/3 in AML drive time-dependent AbSp (intron retention) and splice variant switching, which in MM is induced by culture with BMSC; and conversely, that analogous changes are induced in BMSC cultured with tumor cells. They define role of deregulated expression of the PTBPs in MM/AML pathogenesis, and suggest novel targets for therapeutic intervention.
Stone:Pfizer: Consultancy; Jazz: Consultancy; Fujifilm: Consultancy; Merck: Consultancy; Cornerstone: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Novartis: Consultancy, Research Funding; Sumitomo: Consultancy; Ono: Consultancy; Orsenix: Consultancy; Otsuka: Consultancy; AbbVie: Consultancy; Agios: Consultancy, Research Funding; Amgen: Consultancy; Argenx: Other: Data and Safety Monitoring Board; Arog: Consultancy, Research Funding; Astellas: Consultancy. Griffin:Astellas Pharma: Consultancy; Novartis Pharma: Other: Grant, Patents & Royalties: Royalties ; Analysis Group: Consultancy; Sun Pharmaceuticals: Consultancy; RXi Pharmaceuticals: Consultancy; Lilly Pharmaceuticals: Other: Grant; Myeloproliferative Neoplasia Foundation: Other: Grant . Anderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy.
Multiple Myeloma (MM) is a disease driven by numerous genetic and epigenetic alterations, however true drivers of the disease have yet to be identified.
To identify new dependencies and actionable ...therapeutic targets in MM we integrated gene expression and genetic dependency (CRISPR KO).
We found that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells (NPC). Loss-of-function studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1 confirmed significant impact on MM cell viability. Transcriptomic analysis by RNA-sequencing revealed a small set of genes commonly modulated in MM cell lines upon BOB-1 depletion, including the XBP1- BHLHA15 axis involved in lipid synthesis and unfolded protein response (UPR). Interestingly, among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 (HMOX1), that was affected via the NRF2/Keap1 pathway. We observed that HMOX1 expression is significantly lower in MM cells from patients compared to normal plasma cells and correlates with poor clinical outcome, suggesting important role in MM. Moreover, we found that siRNA depletion of HMOX1 reverted the inhibition of MM cell growth caused by BOB-1 KD, confirming significant role for HMOX1 in the BOB-1 addiction observed in MM cells. Next, we performed gene set enrichment analysis (GSEA) and observed ribosome biogenesis pathways and mRNA translation and elongation processes, along with WNT and senescence pathways, to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. In MM cell lines, BOB-1 knockdown decreased de novo protein synthesis, while its overexpression significantly enhances protein synthesis compared to control cells. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular kappa and lambda light chains production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result, BOB-1 depletion was associated with induction of resistance to proteasome inhibition.
In conclusion, we report BOB1 as a specific dependency in MM cells with potential role on modulating the protein load/capacity balance in MM cells and therefore the sensitivity to proteasome inhibition.
Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34+ bone marrow cells from normal donors. ...Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.
•Overall, our results suggest that NOTCH2 and FLT3 aberrant splicing is a common event in AML that correlates with disease status and may correlate with disease outcomes.•Selected variants of NOTCH2 and FLT3 transcripts were detected in a significant number of AML patients and could be useful as disease markers.
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