A phase II trial of an alternative schedule of palbociclib and embedded serum TK1 analysis

Palbociclib 3-weeks-on/1-week-off, combined with hormonal therapy, is approved for hormone receptor positive (HR+)/HER2-negative (HER2-) advanced/metastatic breast cancer (MBC). Neutropenia is the most frequent adverse event (AE). We aim to determine whether an alternative 5-days-on/2-days-off weekly schedule reduces grade 3 and above neutropenia (G3 + ANC) incidence. In this single-arm phase II trial, patients with HR+/HER2- MBC received palbociclib 125 mg, 5-days-on/2-days-off, plus letrozole or fulvestrant per physician, on a 28-day cycle (C), as their first- or second-line treatment. The primary endpoint was G3 + ANC in the first 29 days (C1). Secondary endpoints included AEs, efficacy, and serum thymidine kinase 1 (sTK1) activity. At data-cutoff, fifty-four patients received a median of 13 cycles (range 2.6-43.5). The rate of G3 + ANC was 21.3% (95% CI: 11.2-36.1%) without G4 in C1, and 40.7% (95% CI: 27.9-54.9%), including 38.9% G3 and 1.8% G4, in all cycles. The clinical benefit rate was 80.4% (95% CI: 66.5-89.7%). The median progression-free survival (mPFS) (95% CI) was 19.75 (12.11-34.89), 33.5 (17.25-not reached [NR]), and 11.96 (10.43-NR) months, in the overall, endocrine sensitive or resistant population, respectively. High sTK1 at baseline, C1 day 15 (C1D15), and C2D1 were independently prognostic for shorter PFS (p = 9.91 × 1

Genomic complexity predicts resistance to endocrine therapy and CDK4/6 inhibition in hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer

PURPOSE: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance. EXPERIMENTAL DESIGN: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB). RESULTS: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P \u3c 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients. CONCLUSIONS: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence

Loss of SrbB attenuates <i>Aspergillus fumigatus</i> virulence through reductions in pulmonary fungal burden.

<p>(A). 6–8 week old immunosuppressed CD-1 mice (each group N = 20) inoculated via the intranasal route with 2×10⁶ conidia of wild type, Δ<i>srbB</i> and <i>srbB</i>-reconstituted strains. Comparing wild type and reconstituted strain Kaplan-Meier curves with the Δ<i>srbB</i> strain shows a significant increase in survival for the animals inoculated with the Δ<i>srbB</i> strain (Log rank test, p = 0.0027). All control PBS inoculated animals survived. (B) Triamcinolone mouse model was used for fungal burden analysis. Mice were infected with 10⁶ conidia of each strain and lungs were collected on day +3. Data represented are the mean and standard error of 3–5 mice per group.</p

Selected SrbA ChIP Peaks with flanking gene information.

<p>Selected SrbA ChIP Peaks with flanking gene information.</p

RNA-seq and nCounter Analyses of Δ<i>srbA</i> Confirms SrbA Regulation of ChIP-seq Target Genes <i>in vitro</i> and <i>in vivo</i> during Invasive Pulmonary Aspergillosis.

<p>(A). Enrichment of RNA-seq differentially expressed genes in GO/Funcat categories of up- and down-regulated genes in srbA cells under hypoxia 120 minutes versus WT (B). Analysis of transcript levels of 12 of the ChiP-seq target genes <i>in vivo</i> in a murine model of invasive pulmonary aspergillosis for wild type (CEA10) and <i>in vitro</i> under normoxic/hypoxic conditions for Δ<i>srbA</i> and wild type <i>in vivo</i> samples were at 48–96 hours post-infection (grey, n = 16). <i>in vitro</i> samples were wild type normoxia (red, n = 2) and hypoxia (blue, n = 6) followed by Δ<i>srbA</i> under normoxia (red, n = 2) and hypoxia (blue, n = 6). Time under hypoxia for both wild type and Δ<i>srbA</i> ranged from 30 to 120 minutes. Expression values are represented as total number of normalized counts per transcript. Quantitation and normalization was as follows: Digital counts for 60 genes (ChIP targets, housekeeping genes and other genes of interest) were adjusted for binding efficiency with background subtraction using the included positive and negative controls from the manufacturer as per NanoString nCounter data analysis guidelines. Data sets were normalized to facilitate across sample comparisons using the geometric mean of 20 stably expressed genes.</p

Restoration of <i>srbB</i> transcript levels in Δ<i>srbA</i> promotes growth in hypoxia.

<p>(A). Using two promoters, <i>flavA</i>(p) and <i>gpdA</i>(p), <i>srbB</i> was expressed in Δ<i>srbA</i>. Expression levels of <i>srbB</i> in TDC43.18 (<i>flavA</i>(p)) and TDC44.2 (<i>gpdA</i>(p)) in normoxia or hypoxia 2 h were verified using quantitative PCR. Data are presented as the mean and standard error. Compared to Δ<i>srbA</i>, <i>srbB</i> transcript in TDC43.18 and TDC44.2 was more abundant by 5- and 3.7-fold in normoxia and 2.7- and 1.4-fold in hypoxia 2 h, respectively. This indicates these promoters function properly. (B). Sequentially diluted conidia (10²–10⁵) were inoculated on GMM plates and culture in normoxia or hypoxia at 37°C for 3 days. Increased expression of <i>srbB</i> using <i>flavA</i>(p) or <i>gpdA</i>(p) partially restores defective hypoxia growth in Δ<i>srbA</i>. (C). Susceptibility of TDC43.18 and TDC44.2 to the triazole drug, voriconazole (VCZ) was tested. A million conidia were overlaid on GMM, and VCZ in DMSO was applied to the center of the plate. Cleared areas represent inhibited fungal growth in response to VCZ. Although growth in hypoxia was partially rescued in both strains as shown (B), increased susceptibility to VCZ in Δ<i>srbA</i> compared to wild type was not affected by restoration of <i>srbB</i> expression.</p

SrbB is a transcriptional regulator of genes involved in carbon metabolism, lipid metabolism, and heme biosynthesis.

<p>The FungiFun2 web server was utilized to assign FunCat and gene ontology enrichment in genes with transcript levels increased or decreased 4 fold in Δ<i>srbB</i> compared to the wild type strain. Statistically significant (P≤0.05) FunCat categories are presented at the respective time points in hypoxia.</p

Binding of SrbB to the promoter of specific SrbA target genes and binding of SrbA in Δ<i>srbB</i>.

<p>(A) SrbB tagged with GFP was expressed in <i>A. fumigatus</i> wild type. The resulting strain was cultured in normoxia at 37°C, 250 rpm for 18 hours and shifted to hypoxia for additional incubation for 4 hours. ChIP was conducted using GFP antibody followed by ChIP-qPCR to study SrbB enrichment on the promoters of SrbA target genes. Compared to wild type control, SrbB enrichment was significant in SrbB:GFP for <i>srbA</i>, <i>srbB</i>, <i>erg25A</i>, <i>hem13</i>, and <i>alcC</i>, which suggest SrbB directly binds on the promoter of these genes for transcriptional regulation. In contrast, SrbB enrichment on the promoters of <i>erg11A</i> and <i>actA</i> were not significant. Data are presented as the mean and standard error of two biological replicates, and analyzed by two-way ANOVA followed by Bonferroni posttest. (B) SrbA binding to the promoter of <i>srbA</i>, <i>srbB</i>, and <i>erg11A</i> in Δ<i>srbB</i> was examined by ChIP-qPCR. Compared to wild type, SrbA enrichment on the gene promoters was not altered by disruption of SrbB. Data are presented as the mean and standard error of two biological replicates and analyzed by two-way ANOVA followed by Bonferroni posttests.</p

Co-Regulation of SrbA target genes by SrbB.

<p>Conidia of each strain were cultured in normoxia at 37°C, 250 rpm for 18 hours and shifted to hypoxia for additional incubation for 4 hours. Expression of SrbA target genes involved in ergosterol biosynthesis, nitrate assimilation, heme biosynthesis, and carbohydrate metabolism in the strains was studied using qRT-PCR. Data are presented as the mean and standard error of two biological replicates, and analyzed by one-way ANOVA followed by Bonferroni's posttests. Expression of <i>erg1</i>, <i>erg25A</i>, <i>niiA</i>, <i>hem13</i>, and <i>alcC</i> requires both SrbA and SrbB. In contrast, expression of <i>erg3B</i>, <i>erg11A</i>, and <i>niaD</i> are regulated by only SrbA. SrbB appears to have a dominant role over SrbA in regulation of <i>hem13</i> and <i>alcC</i> expression. ΔΔ<i>srbAsrbB</i>: a double knock-out mutant of <i>srbA</i> and <i>srbB</i>, <i>srbB</i>-ove;Δ<i>srbA</i>: Δ<i>srbA</i> with restored <i>srbB</i> expression, <i>srbA</i>-ove;Δ<i>srbB</i>: a <i>srbA</i> overexpression strain in Δ<i>srbA</i>Δ<i>srbB</i>.</p