Immunologic Profiling of Immune-Related Cutaneous Adverse Events with Checkpoint Inhibitors Reveals Polarized Actionable Pathways

Purpose: Immune-related cutaneous adverse events (ircAEs) occur in ≥50% of patients treated with checkpoint inhibitors (CPI), but mechanisms are poorly understood. Experimental Design: Phenotyping/biomarker analyses were conducted in 200 patients on CPIs (139 with ircAEs, 61 without, control) to characterize their clinical presentation and immunologic endotypes. Cytokines were evaluated in skin biopsies, skin tape strip (STS) extracts and plasma using real-time PCR and Meso Scale Discovery multiplex cytokine assays. Results: Eight ircAE phenotypes were identified: pruritus (26%), maculopapular rash (MPR; 21%), eczema (19%), lichenoid (11%), urticaria (8%), psoriasiform (6%), vitiligo (5%), and bullous dermatitis (4%). All phenotypes showed skin lymphocyte and eosinophil infiltrates. Skin biopsy PCR revealed the highest increase in IFN-gamma mRNA in patients with lichenoid (p<0.0001) and psoriasiform dermatitis (p<0.01) as compared to patients without ircAEs, while the highest IL-13 mRNA levels were detected in the eczema (p<0.0001, compared to control). IL-17A mRNA was selectively increased in psoriasiform (p<0.001), lichenoid (p<0.0001), bullous dermatitis (p<0.05) and MPR (p<0.001), compared to control. Distinct cytokine profiles were confirmed in STS and plasma. Analysis determined increased skin/plasma IL-4 cytokine in pruritus, skin IL-13 in eczema, plasma IL-5 and IL-31 in eczema and urticaria, and mixed-cytokine pathways in MPR. Broad inhibition via corticosteroids or type 2-cytokine targeted inhibition resulted in clinical benefit in these ircAEs. In contrast, significant skin upregulation of type 1/type 17 pathways was found in psoriasiform, lichenoid, bullous dermatitis, and type 1 activation in vitiligo. Conclusions: Distinct immunologic ircAE endotypes suggest actionable targets for precision medicine-based interventions

A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during Drosophila eye patterning.

Ubiquitination is a crucial post-translational modification that can target proteins for degradation. The E3 ubiquitin ligases are responsible for recognizing substrate proteins for ubiquitination, hence providing specificity to the process of protein degradation. Here, we describe a genetic modifier screen that identified E3 ligases that modified the rough-eye phenotype generated by expression of cindrRNAi transgenes during Drosophila eye development. In total, we identified 36 E3 ligases, as well as 4 Cullins, that modified the mild cindrRNA mis-patterning phenotype. This indicates possible roles for these E3s/Cullins in processes that require Cindr function, including cytoskeletal regulation, cell adhesion, cell signaling and cell survival. Three E3 ligases identified in our screen had previously been linked to regulating JNK signaling

The <i>cindr</i>^<i>RNAi</i> eye is modified by JNK activity.

<p>(A) Eye of a <i>GMR-GAL4</i> heterozygote and (B) <i>GMR>cindr</i>^<i>RNAi</i> adult. (C) <i>cindr</i>^<i>RNAi</i>–induced mis-patterning was mildly enhanced by ectopic <i>bsk</i> (D) but (D) on its own <i>bsk</i> expression did not disrupt the eye. Similarly (E) ectopic <i>slpr</i> enhanced the <i>cindr</i>^<i>RNAi</i> rough eye but (F) the <i>GMR>slpr</i> adult eye was correctly formed. (G) <i>puc</i>^<i>H246</i>, (H) <i>bsk</i>^<i>1</i> and (I) ectopic <i>puc</i> enhanced <i>cindr</i>^<i>RNAi</i>- mis-patterning, whilst (J) expression of only <i>puc</i> did not perturb patterning. Similarly (K) <i>Traf4</i>^{<i>EY0977</i>1} enhanced the <i>GMR>cindr</i>^<i>RNAi</i> rough eye whilst (L) <i>GMR>Traf4</i>^{<i>EY09771</i>} adults had correctly patterned eyes. (M) <i>Uev1a</i>^{<i>DG14805</i>} severely enhanced the <i>GMR>cindr</i>^<i>RNAi</i> rough eye.</p

A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during <i>Drosophila</i> eye patterning

<div><p>Ubiquitination is a crucial post-translational modification that can target proteins for degradation. The E3 ubiquitin ligases are responsible for recognizing substrate proteins for ubiquitination, hence providing specificity to the process of protein degradation. Here, we describe a genetic modifier screen that identified E3 ligases that modified the rough-eye phenotype generated by expression of <i>cindr</i>^<i>RNAi</i> transgenes during <i>Drosophila</i> eye development. In total, we identified 36 E3 ligases, as well as 4 Cullins, that modified the mild <i>cindr</i>^<i>RNA</i> mis-patterning phenotype. This indicates possible roles for these E3s/Cullins in processes that require Cindr function, including cytoskeletal regulation, cell adhesion, cell signaling and cell survival. Three E3 ligases identified in our screen had previously been linked to regulating JNK signaling.</p></div

Modification of the rough-eye phenotype by alleles of E3 ligases linked to JNK signaling.

<p>(A) A correctly patterned heterozygous <i>GMR-GAL4/+</i> eye. (B) Mild mis-patterning manifested as mildly disordered facets that were not arranged in straight rows in the <i>GMR>cindr</i>^<i>RNAi</i> eye. Mis-patterning was suppressed by (C) ectopic <i>nopo</i> (<i>nopo</i>^<i>G5845</i>) but (D) on its own, <i>nopo</i> expression did not disrupt the eye. (E) <i>park</i>^{<i>c00062</i>} enhanced <i>cindr</i>^<i>RNAi</i> mis-patterning whilst (F) <i>park</i>^<i>1</i> and (G) <i>park</i>^Δ21 suppressed <i>cindr</i>^<i>RNAi</i> mis-patterning. (H) <i>Traf6</i>^<i>EP325</i> and (I) <i>Traf6</i>^{<i>EP1516</i>} also modestly suppressed the <i>cindr</i>^<i>RNAi</i> rough eye. (J) <i>Traf6</i>^<i>EP325</i> and (K) <i>Traf6</i>^{<i>EP1516</i>} did not disrupt the eye when crossed to <i>GMR-GAL4</i>.</p

Quantification of patterning defects in retinas dissected at 40 h APF.

<p>Quantification of patterning defects in retinas dissected at 40 h APF.</p

List of E3 ligase and Cullin proteins tested in screen.

<p>List of E3 ligase and Cullin proteins tested in screen.</p

Patterning of the pupal retinal was modified by interactions between <i>park</i>, <i>nopo</i> and <i>Traf6</i> and <i>cindr</i>.

<p>(A) A single ommatidium in a wild-type eye dissected at 40 h APF, with constituent cell types indicated. Photoreceptors are positioned beneath the surface of the tissue and not clearly observed in this image of the apical eye surface. (B) Small region of a correctly patterned control pupal retina and (C) retina expressing <i>cindr</i>^<i>RNAi</i> together with <i>lacZ</i> or (D) only <i>cindr</i>^<i>RNAi</i>. Expression of (E) <i>nopo</i>^<i>G5845</i>, (F) <i>nopo</i>^<i>G5845</i> and <i>cindr</i>^<i>RNAi</i>. (G) Expression of <i>cindr</i>^<i>RNAi</i> in a <i>nopo</i>^{<i>excl42</i>} heterozygote and (H) <i>nopo</i>^<i>Z1447</i> heterozygote. Expression of (I) <i>park</i>, (J) <i>park</i> and <i>cindr</i>^<i>RNAi</i>. (K) Expression of <i>cindr</i>^<i>RNAi</i> in a <i>park</i>^<i>1</i> heterozygote and (L) <i>park</i>^Δ21, heterozygote. (M) Expression of <i>Traf6</i>^<i>S</i> and (N) <i>Traf6</i>^<i>S</i> and <i>cindr</i>^<i>RNAi</i>. Anti-ECad was used to visualize all adherens junctions of retinas. Fluorescence images have been transformed into greyscale and interommatidial cells pseudo-colored red in order to highlight the honeycomb lattice. Examples of patterning defects are indicated as follows: blue arrow = mis-orientation of ommatidial core; outlined in green = small primary pigment cells; yellow circle = tertiary position not defined; blue circle = bristle misplaced and star-like arrangement of cells around bristle; orange asterisks = two cells rather than one in a secondary pigment cell position; blue asterisks = cells grouped in multiple rows rather than single file.</p

A screen for E3 ligases that regulate <i>Drosophila</i> eye development.

<p>(A) An eye of the Canton S strain of wild type flies. (B) Cartoon drawing of columnar adult ommatidia. A bundle of photoreceptor cells (grey) forms the core of each ommatidium. These are surrounded by epithelial pigment cells (dark pink). Each ommatidium is capped with a lens (light grey). (C) The eye of an adult heterozygous for <i>GMR-GAL4</i>. The eye is wild type in appearance. (D) The eye of an adult heterozygous for <i>GMR-GAL4</i> and <i>UAS-cindr</i>^<i>RNAi</i>. The eye is mildly mis-patterned. (E) Crossing scheme used in screen. (F) Mis-patterned eye of a fly heterozygous for <i>cbl</i>^<i>F165</i> and expressing <i>cindr</i>^<i>RNAi</i>. (G) The correctly-patterned eye of an adult heterozygous for <i>cbl</i>^<i>F165</i> and <i>GMR-GAL4</i>.</p

Summary of E3 ligase loci and Cullins identified and included in screen.

<p>Summary of E3 ligase loci and Cullins identified and included in screen.</p