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T-cell infiltration for patient LH-01-26 with BRAF V600 mutation in comparison to study population

Multispectral immunofluorescence. Number of cells positive for exhaustion (PD-L1, TIM3, LAG3, CTLA4) and activation markers (OX40) in baseline tumor specimens (A); Quantitation of signals per specimen (B). There are more PDL1 positive tumor cell in the pre-treatment metastatic site biopsy. While there are more activated, partially activated T cell-1 and exhausted T cell-1 in the primary tumor specimens (*p

Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors has emerged as a major obstacle that hinders their utility beyond ER+ breast cancer. In this study, CDK4/6-dependent and -resistant models were employed to identify functional determinants of response to pharmacologic CDK4/6 inhibitors. In all models tested, the activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity. While depleting CDK4 and 6 was sufficient to limit proliferation in specific resistance settings, RB loss rendered cells completely independent of these kinases. The main downstream target in this context was the activation status of CDK2, which was suppressed with CDK4/6 inhibition in an RB-dependent fashion. Protein levels of p27 were associated with plasticity/rigidity of the cell cycle and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via inhibition of SKP2 and targeting the MEK/ERK pathway enhanced the cytostatic effect of CDK4/6 inhibitors. Mice bearing ER+ xenografts displayed a durable antitumor response to palbociclib; however, over the course of treatment, few cells retained RB phosphorylation, which was associated with limited p27 protein levels as determined by multispectral imaging. Similarly, combination treatment of palbociclib with a MEK inhibitor in pancreatic cancer PDX models upregulated p27 and further enhanced the in vivo tumor response to palbociclib. Collectively, these results suggest that the cell cycle plasticity, which enables tumor models to evade palbociclib-mediated activation of RB, could be targeted using a clinically applicable CDK2 inhibitor.Significance:This work provides a mechanistic insight toward understanding the functional roles of multiple cell cycle regulators that drive plasticity and sensitivity to CDK4/6 inhibition.

Supplementary information

Consort Diagram.

Intratumoral NK cells (baseline) by change in tumor size (A) and change in CEA (B).

Correlate of baseline and on-treatment changes in intratumoral immune cells (flow cytometry) with clinical endpoints (Supplementary Table 2A), immune checkpoint expression by multispectral immunofluorescence in tumor microenvironment (Supplementary Table 2B) and correlation with clinical endpoints (Supplementary Table 2C), immune cells (flow cytometry) in peripheral blood samples at baseline and on treatment (Supplementary Table 2D) and correlation with clinical outcomes (Supplementary Table 2E).

Changes in peripheral blood immune cell populations on treatment.

Purpose:We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC).Patients and Methods:In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence.Results:Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9–5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01).Conclusions:The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.

Adverse events with maximum grade seen

Change in CEA over time.

The retinoblastoma tumor suppressor gene (RB1) plays a critical role in coordinating multiple pathways that impact cancer initiation, disease progression, and therapeutic responses. Here we probed molecular features associated with the RB-pathway across 31 tumor-types. While the RB-pathway has been purported to exhibit multiple mutually exclusive genetic events, only RB1 alteration is mutually exclusive with deregulation of CDK4/6 activity. An ER+ breast cancer model with targeted RB1 deletion was used to identify signatures of CDK4/6 activity and RB-dependency (CDK4/6-RB integrated signature). This signature was prognostic in tumor-types with gene expression features indicative of slower growth. Single copy loss on chromosome 13q encompassing the RB1 locus is prevalent in many cancers, yielding reduced expression of multiple genes in cis, and is inversely related to the CDK4/6-RB integrated signature supporting a cause-effect relationship. Genes that are positively and inversely correlated with the CDK4/6-RB integrated signature define new tumor-specific pathways associated with RB-pathway activity., Erik Knudsen et al. present a pan-cancer analysis of the RB tumor suppressor protein pathway in 31 tumor types. They find that pathway deregulation is multi-faceted with context dependent association with survival. However, gene expression features are surprisingly invariant and support new therapeutic targets.

Purpose: We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC). Patients and Methods: In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence. Results: Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9–5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01). Conclusions: The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease.

Progression through G1/S phase of the cell cycle is coordinated by cyclin-dependent kinase (CDK) activities. Here, we find that the requirement for different CDK activities and cyclins in driving cancer cell cycles is highly heterogeneous. The differential gene requirements associate with tumor origin and genetic alterations. We define multiple mechanisms for G1/S progression in RB-proficient models, which are CDK4/6 independent and elicit resistance to FDA-approved inhibitors. Conversely, RB-deficient models are intrinsically CDK4/6 independent, but exhibit differential requirements for cyclin E. These dependencies for CDK and cyclins associate with gene expression programs that denote intrinsically different cell-cycle states. Mining therapeutic sensitivities shows that there are reciprocal vulnerabilities associated with RB1 or CCND1 expression versus CCNE1 or CDKN2A. Together, these findings illustrate the complex nature of cancer cell cycles and the relevance for precision therapeutic intervention.

Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors has emerged as a major obstacle that hinders their utility beyond ER+ breast cancer. In this study, CDK4/6-dependent and -resistant models were employed to identify functional determinants of response to pharmacologic CDK4/6 inhibitors. In all models tested, the activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity. While depleting CDK4 and 6 was sufficient to limit proliferation in specific resistance settings, RB loss rendered cells completely independent of these kinases. The main downstream target in this context was the activation status of CDK2, which was suppressed with CDK4/6 inhibition in an RB-dependent fashion. Protein levels of p27 were associated with plasticity/rigidity of the cell cycle and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via inhibition of SKP2 and targeting the MEK/ERK pathway enhanced the cytostatic effect of CDK4/6 inhibitors. Mice bearing ER+ xenografts displayed a durable antitumor response to palbociclib; however, over the course of treatment, few cells retained RB phosphorylation, which was associated with limited p27 protein levels as determined by multispectral imaging. Similarly, combination treatment of palbociclib with a MEK inhibitor in pancreatic cancer PDX models upregulated p27 and further enhanced the in vivo tumor response to palbociclib. Collectively, these results suggest that the cell cycle plasticity, which enables tumor models to evade palbociclib-mediated activation of RB, could be targeted using a clinically applicable CDK2 inhibitor. Significance: This work provides a mechanistic insight toward understanding the functional roles of multiple cell cycle regulators that drive plasticity and sensitivity to CDK4/6 inhibition.

ObjectiveThis study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer.DesignPatient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment.ResultsWe found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors.ConclusionsTogether, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy.

PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data.

We investigated a small outbreak of varicella in a long-term-care facility after a case of herpes zoster. Clinical specimens and environmental samples were collected from all case patients and from surfaces in the case patients' rooms and other common-use areas. Wild-type varicella-zoster virus (VZV) DNA was identified in all 3 varicella case patients, and high concentrations of VZV DNA were detected in environmental samples from the room of the herpes zoster case patient. Genotypic analysis showed that the identical VZV strain was present in all samples; moreover, the strain was a unique Mosaic genotype isolate that included a stable Oka vaccine marker that had hitherto never been observed in a wild-type strain of VZV. This study provides evidence for the value of including environmental sampling during the investigation of varicella outbreaks and illustrates the importance of evaluating multiple vaccine-associated markers for the discrimination of vaccine virus from wild-type VZV.

In a limited area of sympatry in New York State, hybridization occurs between Prosimulium multidentatum (Twinn) and “forms” 2 and 3 of P. magnum Dyar and Shannon. Salivary gland chromosomes of the interspecific hybrids are the composite of those of the parental species with respect to fixed inversion differences, sex chromosomes and chromocenter attachment of centromeres. Backcross hybrids with P. multidentatum occur in the same area. The preferred direction of cross (P. multidentatum ♀ x P. magnum ♂) is rationalized by the earlier emergence of P. multidentatum, and the earlier emergence of males and greater longevity of females of both species. Chromosome pairing, both in F1 and backcross, is loose in hybrid segments and tight in species homozygous parts. Likewise chiasma frequency is low in hybrid as compared to parentally homozygous chromosomes. In the face of locally recurrent hybridization species integrity is maintained by meiotic irregularities in the hybrids and especially by disturbances in the sex chromosome balance of backcross hybrids, the sex chromosomes of P. multidentatum and P. magnum being non-homologous.